Dna sequencing reaction additive

ABSTRACT

The present invention provides methods, compositions, mixtures and kits utilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencing reactions, and in particular, sequencing reactions where deoxynucleoside triphosphates comprising a 3′-O position capped by a disulfide-based 3′-terminator group are used. In one embodiment, the deoxynucleoside triphosphates comprise a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. In addition, thiol-containing compounds and scavengers of thio-containing compounds are described. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.

SEQUENCING ADDITIVE

This application is a Continuation of, and claims priority to,co-pending U.S. patent application Ser. No. 15/878,633, filed Jan. 24,2018, the contents of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention provides methods, compositions, mixtures and kitsutilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencingreactions, and in particular, sequencing reactions where deoxynucleosidetriphosphates comprising a 3′-O position capped by a disulfide-based3′-terminator group are used. Such compounds provide new possibilitiesfor future sequencing technologies, including but not limited toSequencing by Synthesis (SBS), including automated SBS in a flow cell.The present invention also provides methods, compositions, mixtures andkits utilizing thiol-containing compounds or derivatives (alternativelymercaptol analogues) as cleave agents of the disulfide based linkers and3′-disulfide capping group of nucleotides.

BACKGROUND OF THE INVENTION

DNA sequencing is one of the most important analytical methods in modernbiotechnology. Detailed reviews on current sequencing technologies areprovided in M. L. Metzker, Nature Reviews 2010, 11, 31 [1], and C. W.Fuller et al., Nature Biotechnology 2009, 27, 1013 [2].

A well-known sequencing method is the Sequencing-by-synthesis (SBS)method. According to this method, the nucleoside triphosphates arereversibly blocked by a 3′OH-protecting group, in particular esters andethers. Examples for esters are alkanoic esters like acetyl, phosphatesand carbonates. The nucleoside triphosphate usually comprises a label atthe base.

A method of enzymatically synthesizing a polynucleotide of apredetermined sequence in a stepwise manner using reversibly3′OH-blocked nucleoside triphosphates was described by Hiatt and Rose(U.S. Pat. No. 5,990,300) [3]. They disclose besides esters, ethers,carbonitriles, phosphates, phosphoramides, carbonates, carbamates,borates, sugars, phosphoramidates, phenylsulfenates, sulfates andsulfones also nitrates as cleavable 3′OH-protecting group. Thedeprotection may be carried out by chemical or enzymatic means. Thereare neither synthesis procedures nor deprotection conditions andenzymatic incorporation data disclosed for the nitrate group. Theclaimed deblocking solution preferably contains divalent cations likeCo2+ and a biological buffer like Tris. 3′OH-blocked nucleosidetriphosphates containing a label are not disclosed.

Buzby (US 2007-0117104) [4] discloses nucleoside triphosphates for SBSwhich are reversibly protected at the 3′-hydroxyl group and carry alabel at the base. The label is connected via a cleavable linker such asa disulfide linker or a photocleavable linker. The linker consists of upto about 25 atoms. The 3′OH-protection group can be besideshydroxylamines, aldehydes, allylamines, alkenes, alkynes, alcohols,amines, aryls, esters, ethers, carbonitriles, phosphates, carbonates,carbamates, borates, sugars, phosphoramidates, phenylsulfanates,sulfates, sulfones and heterocycles also nitrates.

Improvements are needed in order to achieve longer read length andbetter accuracy in nucleic acid sequencing.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, mixtures and kitsutilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencingreactions, and in particular, sequencing reactions where deoxynucleosidetriphosphates comprising a 3′-O position capped by a disulfide-based3′-terminator group are used. Such compounds provide new possibilitiesfor future sequencing technologies, including but not limited toSequencing by Synthesis (SBS), including automated SBS in a flow cell.The present invention provides methods, compositions, mixtures and kitsutilizing deoxynucleoside triphosphates comprising a 3′-O positioncapped by a group comprising methylenedisulfide as a cleavableprotecting group and a detectable label reversibly connected to thenucleobase of said deoxynucleoside. In one embodiment, the presentinvention contemplates a nucleotide analogue with a reversibleprotecting group comprising methylenedisulfide and a cleavableoxymethylenedisulfide linker between the label and nucleobase. Thepresent invention also provides methods, compositions, mixtures and kitsutilizing thiol derivatives (alternatively mercaptol analogues) ascleave agents of the disulfide based linkers and 3′-disulfide cappinggroup of nucleotides pertinant to DNA sequencing technologies (and othertechnologies such as protein engineering) where the disulfide reductionor cleavage step is necessary. In one embodiment, the cleave agent iskitted as a solid powder add-on that the customer will add to the cleavepre-mix buffer according to sequencing workflow instructions. Moreover,the present invention provides embodiments where the thiol-containingcompounds are scavenged with an oxidative wash step. However, apreferred scavenger is cystamine.

In one embodiment, the present invention contemplates mixing polymerase,a plurality of nucleotide analogues, and5-Chloro-2-methyl-4-isothiazolin-3-one so as to create a mixture insolution. In one embodiment, said mixture in solution comprises one ormore additional compounds. In one embodiment, said mixture in solutionis used in an extension reaction, where polymerase incorporatesnucleotides and/or nucleotide analogues into a hybridized primer.

In one embodiment, the present invention contemplates a method ofincorporating nucleotides, comprising: a) providing i) a plurality ofnucleic acid primers and template molecules, and ii) polymerase, aplurality of nucleotide analogues, and5-Chloro-2-methyl-4-isothiazolin-3-one; b) hybridizing at least aportion of said primers to at least a portion of said template moleculesso as to create hybridized primers; and c) exposing said hybridizedprimers to said polymerase and nucleotide analogues in the presence ofsaid 5-Chloro-2-methyl-4-isothiazolin-3-one, under conditions such atleast one nucleotide analogue is incorporated into at least a portion ofsaid hybridized primers so as to create extended primers comprising anincorporated nucleotide analogue. In one embodiment, said5-Chloro-2-methyl-4-isothiazolin-3-one is in solution with2-Methyl-4-isothiazolin-3-one. While it is not intended that the methodbe limited to particular nucleotide analogues, preferred nucleotideanalogues comprise a 3′-O position capped by a disulfide-based3′-terminator group. It is not intended that the method be limited bythe nature of the disulfide-based 3′-terminator group, however apreferred group comprises methylenedisulfide as a cleavable protectinggroup. In one embodiment, said incorporated nucleotide analogue furthercomprises a label attached through a cleavable disulfide linker to thebase. In one embodiment, the method further comprises d) detecting saidlabel of a first labeled nucleotide analogue. In one embodiment, themethod further comprises e) cleaving said linker with a cleaving reagentso as to remove said label. In one embodiment, said cleaving reagentalso removes said disulfide-based 3′-terminator group. A variety ofcleaving reagents are contemplated; however, preferred cleaving reagentsare thiol-containing compounds. In one embodiment, the thiol-containingcompound is a vicinal dithiol-based compound. In one embodiment, thevicinal dithiol-based compound is di-mercaptopropanesulfonate.

The present invention contemplates in another embodiment a method fordetecting labeled nucleotides in a nucleic acid sequence comprising thesteps of a) providing i) nucleic acid template and primer capable ofhybridizing to said template so as to form a primer/templatehybridization complex, ii) polymerase, a plurality of nucleotideanalogues, and 5-Chloro-2-methyl-4-isothiazolin-3-one, wherein saidnucleotide analogues comprise a nucleobase and a sugar, said sugarcomprising a cleavable protecting group on the 3′-O, wherein saidcleavable protecting group comprises a disulfide-based 3′-terminatorgroup, and wherein at least a portion of said nucleotide analoguesfurther comprises a first detectable label attached via a cleavabledisulfide-containing linker to the nucleobase; and iii) a cleave reagentcomprising a thiol-containing compound; b) hybridizing at least aportion of said primers to at least a portion of said template moleculesso as to create hybridized primers; and c) exposing said hybridizedprimers to said polymerase, nucleotide analogues and5-Chloro-2-methyl-4-isothiazolin-3-one under conditions such that afirst nucleotide analogue comprising a first detectable label isincorporated into at least a portion of said hybridized primers in apolymerase catalyzed primer extension reaction so as to create extendedprimers comprising an incorporated nucleotide analogue in a modifiedprimer/template hybridization complex; d) detecting said firstdetectable label of said deoxynucleoside triphosphate in said modifiedprimer/template hybridization complex; and e) introducing said cleavereagent under conditions so as to remove said cleavable protecting groupand said detectable label from said modified primer/templatehybridization complex. In one embodiment, the method further comprisesincorporating a second nucleotide analogue comprising a seconddetectable label during a repeat of step c). In one embodiment, saidthiol-containing compound is a vicinal dithiol-based compound. In oneembodiment, the vicinal dithiol-based compound isdi-mercaptopropanesulfonate. In one embodiment, the nucleobase of saidsecond nucleotide analogue is different from the nucleobase of saidfirst nucleotide analogue. In one embodiment, the nucleotide analoguescomprises a mixture of at least 4 differently labeled nucleotideanalogues representing analogs of A, G, C and T or U. In one embodiment,said detecting allows for the determination of the nucleobase of saidincorporated first nucleotide analogue. In one embodiment, saiddisulfide-based 3′-terminator group comprises methylenedisulfide as acleavable protecting group. In one embodiment, said conditions of stepc) further comprises 2-Methyl-4-isothiazolin-3-one. In one embodiment,the method further comprises f) introducing a scavenger that interactswith the cleave reagent. In one embodiment, said scavenger is cystamine.

As noted, the present invention also contemplates compositions andmixtures. In one embodiment, the present invention contemplates acomposition comprising primers hybridized to template in a solution,said solution comprising 5-Chloro-2-methyl-4-isothiazolin-3-one. In oneembodiment, said hybridized primers and template are immobilized. In oneembodiment, hybridized primers and template are in a flow cell. In oneembodiment, said solution further comprises2-Methyl-4-isothiazolin-3-one.

In one embodiment, the present invention contemplates kits comprisingprimers, nucleotide analogues, polymerase and5-Chloro-2-methyl-4-isothiazolin-3-one. In one embodiment, each reagentis in a separate tube. In one embodiment, the kit comprises instructionsto mix said polymerase, nucleotide analogues and5-Chloro-2-methyl-4-isothiazolin-3-one into a solution. In oneembodiment, said solution further comprises2-Methyl-4-isothiazolin-3-one.

In yet another embodiment, the present invention contemplates a methodof incorporating nucleotides, comprising: a) providing i) a plurality ofnucleic acid primers and template molecules, and ii) an extend reagentcomprising polymerase, a plurality of nucleotide analogues, and5-Chloro-2-methyl-4-isothiazolin-3-one (e.g. in solution); b)hybridizing at least a portion of said primers to at least a portion ofsaid template molecules so as to create hybridized primers; and c)exposing said hybridized primers to said extend reagent under conditionssuch that a first nucleotide analogue is incorporated into at least aportion of said hybridized primers so as to create extended primerscomprising an incorporated nucleotide analogue. In one embodiment, saidincorporated nucleotide analogue comprises a 3′-O position capped by adisulfide-based 3′-terminator group. In one embodiment, saiddisulfide-based 3′-terminator group comprises methylenedisulfide as acleavable protecting group. In one embodiment, said incorporatednucleotide analogue further comprises a label attached through acleavable disulfide linker to the base. In one embodiment, said extendreagent further comprises 2-Methyl-4-isothiazolin-3-one. In oneembodiment, the method further comprises d) detecting said label of afirst labeled nucleotide analogue. In one embodiment, the method furthercomprises e) cleaving said linker with a cleaving reagent so as toremove said label. In one embodiment, said cleaving reagent also removessaid disulfide-based 3′-terminator group. In one embodiment, saidcleaving reagent is a thiol-containing compound. In one embodiment, thethiol-containing compound is a vicinal dithiol-based compound. In oneembodiment, the vicinal dithiol-based compound isdi-mercaptopropanesulfonate.

In yet another embodiment, the present invention contemplates a methodfor detecting labeled nucleotides in a nucleic acid sequence comprisingthe steps of a) providing i) nucleic acid template and primer capable ofhybridizing to said template so as to form a primer/templatehybridization complex, ii) an extend reagent comprising polymerase, aplurality of nucleotide analogues, and5-Chloro-2-methyl-4-isothiazolin-3-one, wherein said nucleotideanalogues comprise a nucleobase and a sugar, said sugar comprising acleavable protecting group on the 3′-O, wherein said cleavableprotecting group comprises a disulfide-based 3′-terminator group, andwherein at least a portion of said nucleotide analogues furthercomprises a first detectable label attached via a cleavabledisulfide-containing linker to the nucleobase; and iii) a cleave reagentcomprising a thiol-containing compound; b) hybridizing at least aportion of said primers to at least a portion of said template moleculesso as to create hybridized primers; and c) exposing said hybridizedprimers to said extend reagent under conditions such that a firstnucleotide analogue comprising a first detectable label is incorporatedinto at least a portion of said hybridized primers in a polymerasecatalyzed primer extension reaction so as to create extended primerscomprising an incorporated nucleotide analogue in a modifiedprimer/template hybridization complex; d) detecting said firstdetectable label of said deoxynucleoside triphosphate in said modifiedprimer/template hybridization complex; and e) introducing said cleavereagent under conditions so as to remove said cleavable protecting groupand said detectable label from said modified primer/templatehybridization complex. In one embodiment, the method further comprisesincorporating a second nucleotide analogue comprising a seconddetectable label during a repeat of step c). In one embodiment, thethiol-containing compound is a vicinal dithiol-based compound. In oneembodiment, the vicinal dithiol-based compound isdi-mercaptopropanesulfonate. In one embodiment, the nucleobase of saidsecond nucleotide analogue is different from the nucleobase of saidfirst nucleotide analogue. In one embodiment, said extend reagentcomprises a mixture of at least 4 differently labeled nucleotideanalogues representing analogs of A, G, C and T or U. In one embodiment,said detecting allows for the determination of the nucleobase of saidincorporated first nucleotide analogue. In one embodiment, saiddisulfide-based 3′-terminator group comprises methylenedisulfide as acleavable protecting group. In one embodiment, said extend reagentfurther comprises 2-Methyl-4-isothiazolin-3-one. In one embodiment, themethod further comprises f) introducing a scavenger that interacts withthe cleave reagent. In one embodiment, said scavenger is cystamine.

In terms of mixtures, the present invention in one embodimentcontemplates deoxynucleoside triphosphates comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase and a 3′-Oposition capped by a group comprising methylenedisulfide as a cleavableprotecting group in mixtures with one or more additional sequencingreagents, including but not limited to buffers, polymerases, primers,template and the like. In terms of kits, the present inventioncontemplates in one embodiment a sequencing kit where sequencingreagents are provided together in separate containers (or in mixtures),including deoxynucleoside triphosphates comprising a 3′-O positioncapped by a group comprising methylenedisulfide as a cleavableprotecting group, along with (optionally) instructions for using suchreagents in sequencing. It is not intended that the present invention belimited by the number or nature of sequencing reagents in the kit. Inone embodiment, the kit comprises one or more additional sequencingreagents, including but not limited to buffers, polymerases, primers andthe like.

It is not intended that the present invention be limited to anyparticular polymerase. The present invention contemplates engineered(e.g. mutated) polymerases with enhanced incorporation of nucleotidederivatives. For example, Tabor, S. and Richardson, C. C. ((1995) Proc.Natl. Acad. Sci (USA) 92:6339 [6]) describe the replacement ofphenylalanine 667 with tyrosine in T. aquaticus DNA polymerase and theeffects this has on discrimination of dideoxynucleotides by the DNApolymerase. In one embodiment, the present invention contemplatespolymerases that lack 3′-5′ exonuclease activity (designated exo-). Forexample, an exo-variant of 9° N polymerase is described by Perler etal., 1998 U.S. Pat. No. 5,756,334 [7] and by Southworth et al., 1996Proc. Natl Acad. Sci USA 93:5281 [8]. Another polymerase example is anA486Y variant of Pfu DNA polymerase (Evans et al., 2000. Nucl. Acids.Res. 28:1059 [9]). Another example is an A485T variant of Tsp JDF-3 DNApolymerase (Arezi et al., 2002. J. Mol. Biol. 322:719 [10]). WO2005/024010 A1 relates to the modification of the motif A region and tothe 9° N DNA polymerase, hereby incorporated by reference [11].

In terms of methods, the present invention contemplates both methods tosynthesize deoxynucleoside triphosphates comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase and a 3′-Oposition capped by a group comprising methylenedisulfide as a cleavableprotecting group, as well as methods to utilize deoxynucleosidetriphosphates comprising a 3′-O position capped by a group comprisingmethylenedisulfide as a cleavable protecting group.

In one embodiment, the present invention contemplates, a method fordetecting labeled nucleotides in a DNA sequence comprising the steps ofa) providing a nucleic acid template and primer capable of hybridizingto said template so as to form a primer/template hybridization complex(and in some embodiments, providing it where it has in fact hybridizedtogether), and a cleave reagent comprising a thiol-containing compound;b) adding DNA polymerase and a first deoxynucleoside triphosphate tosaid primer and template so as to create a reaction mixture, said firstdeoxynucleoside triphosphate comprising a nucleobase and a sugar, saidsugar comprising a cleavable protecting group on the 3′-O, wherein saidcleavable protecting group comprises methylenedisulfide, wherein saiddeoxynucleoside triphosphate further comprises a first detectable labelattached via a cleavable oxymethylenedisulfide-containing linker to thenucleobase; c) subjecting said reaction mixture to conditions whichenable a DNA polymerase catalyzed primer extension reaction so as tocreate a modified primer/template hybridization complex, wherein saidfirst deoxynucleoside triphosphate is incorporated (into the primer soas to extend the primer); d) detecting said first detectable label ofsaid deoxynucleoside triphosphate in said modified primer/templatehybridization complex (e.g. imaging); and e) introducing said cleavereagent under conditions so as to remove said cleavable protecting group(e.g. by cleaving it) and (optionally) said detectable label from saidmodified primer/template hybridization complex (e.g. by cleaving thelinker). In one embodiment, the method further comprises adding a seconddeoxynucleoside triphosphate during a repeat of step b), wherein saidsecond deoxynucleoside triphosphate comprises a second detectable label.In one embodiment, the thiol-containing compound is a vicinaldithiol-based compound. In one embodiment, the vicinal dithiol-basedcompound is di-mercaptopropanesulfonate. In one embodiment, saidthiol-containing compound comprises first and second thiol groups. Inone embodiment, said first and second thiol groups are separated by atleast one hydrocarbon atom. In one embodiment, said first and secondthiol groups are separated by a hydrocarbon spacer. In one embodiment,the hydrocarbon spacer comprises at least one functional group. In oneembodiment, said at least one functional group is selected from thegroup consisting of a halogen, an amine, a phosphate, a phosphonate, aphosphoric acid, a carboxylic acid, a sulfoxide and a hydoxyl. In oneembodiment, the nucleobase of said second deoxynucleoside triphosphateis different from the nucleobase of said first deoxynucleosidetriphosphate. In one embodiment, more than 1 labeled nucleotide is used,e.g. a mixture of at least 4 differently labeled, 3′-Omethylenedisulfide capped deoxynucleoside triphosphate compoundsrepresenting analogs of A, G, C and T or U are used in step b). In apreferred embodiment, said detecting allows for the determination of thenucleobase of said incorporated first deoxynucleoside triphosphate.

The present invention contemplates another embodiment of a method fordetecting labeled nucleotides in a DNA sequence comprising the steps ofa) providing a nucleic acid template and primer capable of hybridizingto said template so as to form a primer/template hybridization complex(and in some embodiments, providing it where it is in fact hybridizedtogether), a cleave reagent comprising a thiol-containing compound and acleave scavenger reagent; b) adding DNA polymerase and a firstdeoxynucleoside triphosphate to said primer and template so as to createa reaction mixture, said first deoxynucleoside triphosphate comprising anucleobase and a sugar, said sugar comprising a cleavable protectinggroup on the 3′-O, wherein said cleavable protecting group comprisesmethylenedisulfide, wherein said deoxynucleoside triphosphate furthercomprises a first detectable label attached via a cleavableoxymethylenedisulfide-containing linker to the nucleobase; c) subjectingsaid reaction mixture to conditions which enable a DNA polymerasecatalyzed primer extension reaction so as to create a modifiedprimer/template hybridization complex, wherein said firstdeoxynucleoside triphosphate is incorporated (e.g. into the primer so asto extend the primer); d) detecting said first detectable label of saiddeoxynucleoside triphosphate in said modified primer/templatehybridization complex (e.g. by imaging); e) introducing said cleavereagent under conditions so as to remove said cleavable protecting group(by cleaving) and (optionally) said detectable label from said modifiedprimer/template hybridization complex (by cleaving the linker); and f)introducing said cleave scavenger reagent (e.g. to scavenge any residualcleave reagent). In one embodiment, said thiol-containing compoundcomprises first and second thiol groups. In one embodiment, said firstand second thiol groups are separated by at least one hydrocarbon atom.In one embodiment, said first and second thiol groups are separated by ahydrocarbon spacer. In one embodiment, the hydrocarbon spacer comprisesat least one functional group. In one embodiment, said at least onefunctional group is selected from the group consisting of a halogen, anamine, a phosphate, a phosphonate, a phosphoric acid, a carboxylic acid,a sulfoxide and a hydoxyl. In one embodiment, said cleave reagentcomprises di-mercaptopropanesulfonate. In one embodiment, saiddi-mercaptopropanesulfonate is in a buffer. In one embodiment, saidbuffer is CHES. In one embodiment, the pH of said cleave reagent isbetween 9.0 and 10.0. In a preferred embodiment, said pH of said cleavereagent is 9.5. In one embodiment, said cleave scavenger is an oxidativescavenger. In one embodiment, said oxidative scavenger is hydrogenperoxide. In one embodiment, said oxidative scavenger is ter-butylperoxide. In one embodiment, said hydrogen peroxide is in a buffer. Inone embodiment, said buffer is TRIS. In one embodiment, said TRIS bufferis at a pH of between 8.5 and 9.0. In a preferred embodiment, said TRISbuffer is at a pH of 8.8. In a preferred embodiment, said reactionmixture of step b) is in a flow cell. In one embodiment, said flow cellis positioned on a moving support. In one embodiment, said movingsupport is a rotary stage. In one embodiment, at least a portion of saidflow cell is transparent. In one embodiment, said flow cell isincorporated within an instrument. In one embodiment, the steps of themethod are repeated. In one embodiment, the method further comprisesadding a second deoxynucleoside triphosphate during a repeat of step b),wherein said second deoxynucleoside triphosphate comprises a seconddetectable label attached via a second cleavable oxymethylenedisulfidelinker, wherein said second detectible detectable label is differentfrom said first detectible detectable label. In one embodiment, thethiol-containing compound is a vicinal dithiol-based compound. In oneembodiment, the nucleobase of said second deoxynucleoside triphosphateis different from the nucleobase of said first deoxynucleosidetriphosphate. In one embodiment, a mixture of at least 4 differentlylabeled, 3′-O methylenedisulfide capped deoxynucleoside triphosphatecompounds representing analogs of A, G, C and T or U are used in stepb). In a preferred embodiment, said detecting allows for thedetermination of the nucleobase of said incorporated firstdeoxynucleoside triphosphate.

The present invention, in yet another embodiment, contemplates a methodfor detecting labeled nucleotides in a DNA sequence comprising the stepsof a) providing nucleic acid template and primer capable of hybridizingto said template so as to form a primer/template hybridization complex(and in some embodiments, providing it where it is in fact hybridized),and a flow cell, said flow cell in fluidic communication (e.g. viatubing) with a first reservoir comprising a cleave reagent comprising athiol-containing compound and a second reservoir comprising an oxidativewash; b) adding DNA polymerase and a first deoxynucleoside triphosphateto said primer and template so as to create a reaction mixture in saidflow cell, said first deoxynucleoside triphosphate comprising anucleobase and a sugar, said sugar comprising a cleavable protectinggroup on the 3′-O, wherein said cleavable protecting group comprisesmethylenedisulfide, wherein said deoxynucleoside triphosphate furthercomprises a first detectable label attached via a cleavableoxymethylenedisulfide-containing linker to the nucleobase; c) subjectingsaid reaction mixture to conditions which enable a DNA polymerasecatalyzed primer extension reaction so as to create a modifiedprimer/template hybridization complex, wherein said firstdeoxynucleoside triphosphate is incorporated (e.g. into the primer so asto extend the primer); d) detecting said first detectable label of saiddeoxynucleoside triphosphate in said modified primer/templatehybridization complex (e.g. by imaging); e) introducing said cleavereagent from said first reservoir into said flow cell under conditionsso as to remove said cleavable protecting group (by cleaving) and(optionally) said detectable label from said modified primer/templatehybridization complex (by cleaving the linker); and f) introducing saidoxidative wash from said second reservoir into said flow cell (so as toremove or inactivate any residual cleave reagent). In one embodiment,said thiol-containing compound comprises first and second thiol groups.In one embodiment, said first and second thiol groups are separated byat least one hydrocarbon atom. In one embodiment, said first and secondthiol groups are separated by a hydrocarbon spacer. In one embodiment,the hydrocarbon spacer comprises at least one functional group. In oneembodiment, said at least one functional group is selected from thegroup consisting of a halogen, an amine, a phosphate, a phosphonate, aphosphoric acid, a carboxylic acid, a sulfoxide and a hydoxyl. In oneembodiment, said cleave reagent comprises di-mercaptopropanesulfonate(e.g. in solution). In one embodiment, said di-mercaptopropanesulfonateis in a buffer. In one embodiment, said buffer is CHES. In oneembodiment, the pH of said cleave reagent is between 9.0 and 10.0. Inone embodiment, said pH of said cleave reagent is 9.5. In oneembodiment, said oxidative wash comprises hydrogen peroxide. In oneembodiment, said oxidative wash comprises ter-butyl peroxide. In oneembodiment, said hydrogen peroxide is in a buffer. In one embodiment,said buffer is TRIS.

The present invention also contemplates kits. In one embodiment, thepresent invention contemplates a kit comprising one or more DNAsequencing reagents, instructions and a cleave reagent comprising athiol-containing compound. In one embodiment, said thiol-containingcompound comprises first and second thiol groups. In one embodiment,said first and second thiol groups are separated by at least onehydrocarbon atom. In one embodiment, said first and second thiol groupsare separated by a hydrocarbon spacer. In one embodiment, thehydrocarbon spacer comprises at least one functional group. In oneembodiment, said at least one functional group is selected from thegroup consisting of a halogen, an amine, a phosphate, a phosphonate, aphosphoric acid, a carboxylic acid, a sulfoxide and a hydoxyl. In oneembodiment, the thiol-containing compound is selected from the groupconsisting 2,3-dimercaptopropanesulfonate, 2,3-dimercapto-1-propanol and2,3-dimercaptopropanephosphonate (or salts thereof). In a preferredembodiment, said thiol-containing compound isdi-mercaptopropanesulfonate. In one embodiment, wherein saiddi-mercaptopropanesulfonate is in a buffer. The thiol-containingcompound can be in various forms. In one embodiment, saiddi-mercaptopropanesulfonate is in solid form (e.g. as a powder in atube, vial or other container) in said kit. In one embodiment, the kitfurther comprises a buffer (e.g. in a tube, vial or other container). Inone embodiment, said buffer is CHES. In one embodiment, the kit furthercomprises a cleave scavenger. In a preferred embodiment, said cleavescavenger is an oxidative scavenger. In one embodiment, said oxidativescavenger is hydrogen peroxide. In one embodiment, said oxidativescavenger is ter-butyl peroxide. In one embodiment, said one or more DNAsequencing reagents are selected from the group comprising polymerase,primer, template and nucleotides.

The present invention also contemplates compositions, includingmixtures. In one embodiment, the present invention contemplates acomposition comprising primers hybridized to template in solution, saidsolution comprising a thiol-containing compound. In one embodiment, saidthiol-containing compound comprises first and second thiol groups. Inone embodiment, said first and second thiol groups are separated by atleast one hydrocarbon atom. In one embodiment, said first and secondthiol groups are separated by a hydrocarbon spacer. In one embodiment,the hydrocarbon spacer comprises at least one functional group. In oneembodiment, said at least one functional group is selected from thegroup consisting of a halogen, an amine, a phosphate, a phosphonate, aphosphoric acid, a carboxylic acid, a sulfoxide and a hydoxyl. In oneembodiment, the thiol-containing compound is selected from the groupconsisting 2,3-dimercaptopropanesulfonate, 2,3-dimercapto-1-propanol and2,3-dimercaptopropanephosphonate. In one embodiment, said hybridizedprimers and template are immobilized. In one embodiment, said hybridizedprimers and template are in a flow cell.

The present invention also contemplates flow cells containing solutionsand mixtures. In one embodiment, the present invention contemplates aflow cell, comprising primers hybridized to template in solution, saidsolution comprising a thiol-containing compound and an oxidativescavenger. In one embodiment, said thiol-containing compound comprisesfirst and second thiol groups. In one embodiment, said first and secondthiol groups are separated by at least one hydrocarbon atom. In oneembodiment, said first and second thiol groups are separated by ahydrocarbon spacer. In one embodiment, the hydrocarbon spacer comprisesat least one functional group. In one embodiment, said at least onefunctional group is selected from the group consisting of a halogen, anamine, a phosphate, a phosphonate, a phosphoric acid, a carboxylic acid,a sulfoxide and a hydoxyl. In one embodiment, the thiol-containingcompound is selected from the group consisting2,3-dimercaptopropanesulfonate, 2,3-dimercapto-1-propanol and2,3-dimercaptopropanephosphonate. In one embodiment, said hybridizedprimers and template are immobilized. In one embodiment, said oxidativescavenger is hydrogen peroxide. In one embodiment, said oxidativescavenger is ter-butyl peroxide.

In one embodiment, the invention relates to (a) nucleoside triphosphateswith 3′-O capped by a group comprising methylenedisulfide (e.g. of thegeneral formula —CH₂—SS—R) as cleavable protecting group; and (b) theirlabeled analogs, where labels are attached to the nucleobases viacleavable oxymethylenedisulfide linker (—OCH₂—SS—) (although the linkermay contain additional groups). Such nucleotides can be used in nucleicacid sequencing by synthesis (SBS) technologies. In one embodiment, theinvention relates to the synthesis of nucleotides 3′-O capped by a groupcomprising methylenedisulfide (e.g. —CH₂—SS—R) as cleavable protectinggroup, the deprotection conditions or enzymatic incorporation.

In one embodiment, the invention relates to a deoxynucleosidetriphosphate comprising a cleavable oxymethylenedisulfide linker betweenthe label and nucleobase and a 3′-O capped by a group comprisingmethylenedisulfide as a cleavable protecting group. In one embodiment,the nucleobase of said nucleoside is non-natural. In one embodiment, thenon-natural nucleobase of said nucleoside is selected from the groupcomprising 7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine,and 2-amino adenine. In one embodiment, said group comprisingmethylenedisulfide is —CH₂—SS—R, wherein R is selected from the groupcomprising alkyl and substituted alkyl groups. In one embodiment, saiddetectable label is attached to said nucleobase via cleavableoxymethylenedisulfide linker (e.g. of the formula —OCH₂—SS—). In oneembodiment, said detectable label is a fluorescent label. In oneembodiment, R in the formula (—CH₂—S—S—R) could be alkyl or allyl.

In one embodiment, the invention relates to a deoxynucleosidetriphosphate according to the following structure:

wherein B is a nucleobase and R is selected from the group comprisingalkyl and substituted alkyl groups. In one embodiment, said nucleobaseis a natural nucleobase (cytosine, guanine, adenine, thymine anduracil). In one embodiment, said nucleobase is a non-natural nucleobaseselected from the group comprising 7-deaza guanine, 7-deaza adenine,2-amino,7-deaza adenine, and 2-amino adenine. In the case of analogs,the detectable label may also include a linker section between thenucleobase and said detectable label.

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein B is a nucleobase, R is selected from the group comprising alkyland substituted alkyl groups, and L₁ and L₂ are connecting groups. Inone embodiment, said nucleobase is a natural nucleobase analog. In oneembodiment, said nucleobase is a non-natural nucleobase analog selectedfrom the group comprising 7-deaza guanine, 7-deaza adenine,2-amino,7-deaza adenine, and 2-amino adenine. In the case of analogs,the detectable label may also include a linker section between thenucleobase and said detectable label. In one embodiment, L₁ and L₂ areindependently selected from the group comprising —CO—, —CONH—, —NHCONH—,—O—, —S—, —ON, and —N═N—, alkyl, aryl, branched alkyl, branched aryl orcombinations thereof. It is preferred that L₂ not be “—S—.” In oneembodiment, the present invention contemplates L₁ to be either an amineon the base or a hydroxyl on the base. In one embodiment, said label isselected from the group consisting of fluorophore dyes, energy transferdyes, mass-tags, biotin, and haptenes. In one embodiment, said label isa detectable label.

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein D is selected from the group consisting of disulfide allyl, anddisulfide substituted allyl groups; B is a nucleobase; A is anattachment group; C is a cleavable site core; L₁ and L₂ are connectinggroups; and Label is a label (e.g. a detectable moiety).

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein D is selected from the group consisting of an azide, disulfidealkyl, disulfide substituted alkyl groups; B is a nucleobase; A is anattachment group; C is a cleavable site core; L₁ and L₂ are connectinggroups; and Label is a label. In one embodiment, said nucleobase is anon-natural nucleobase analog selected from the group consisting of7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine, and 2-aminoadenine. In one embodiment, said attachment group A is chemical groupselected from the group consisting of propargyl, hydroxymethyl,exocyclic amine, propargyl amine, and propargyl hydroxyl. In oneembodiment, said cleavable site core is selected from the groupconsisting of:

wherein R₁ and R₂ are independently selected alkyl groups. In oneembodiment, L₁ is selected from the group consisting of —CONH(CH₂)_(x)—,—CO—O(CH₂)_(x)—, —CONH—(OCH₂CH₂O)_(x)—, —CO—O(CH₂CH₂O)_(x)—, and—CO(CH₂)_(x)—, wherein x is 0-10, but more preferably from 1-6. In oneembodiment, L₂ is selected from the group consisting of —NH—,—(CH₂)_(x)—NH—, —C(Me)₂(CH₂)_(x)NH—, —CH(Me)(CH₂)_(x)NH—,—C(Me)₂(CH₂)_(x)CO—, —CH(Me)(CH₂)_(x)CO—,—(CH₂)_(x)OCONH(CH₂)_(y)O(CH₂)_(z)NH—,—(CH₂)_(x)CONH(CH₂CH₂O)_(y)(CH₂)_(z)NH—, and —CONH(CH₂)_(x)—,—CO(CH₂)_(x)—, wherein x, y, and z are each independently selected fromis 0-10, but more preferably from 1-6. In one embodiment, said label isselected from the group consisting of fluorophore dyes, energy transferdyes, mass-tags, biotin, and haptenes. In one embodiment, the compoundhas the structure:

wherein said label is a dye. In one embodiment, the compound has thestructure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the invention relates to a deoxynucleosidetriphosphate according to the following structure:

wherein B is a nucleobase.

In one embodiment, the invention relates to a kit comprising one or moresequencing reagents (e.g. a DNA polymerase) and at least onedeoxynucleoside triphosphate comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase, a 3′-Ocapped by a group comprising methylenedisulfide as a cleavableprotecting group. In one embodiment, said nucleobase is a naturalnucleobase analog. In one embodiment, the nucleobase of said nucleosideis non-natural. In one embodiment, the non-natural nucleobase of saidnucleoside is selected from the group comprising 7-deaza guanine,7-deaza adenine, 2-amino,7-deaza adenine, and 2-amino adenine.

The present invention also contemplates mixtures, i.e. at least onedeoxynucleoside triphosphate comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase, a 3′-Ocapped by a group comprising methylenedisulfide as a cleavableprotecting group in a mixture with one or more additional reagents(whether dry or in solution). In one embodiment, the invention relatesto a reaction mixture comprising a nucleic acid template with a primerhybridized to said template, a DNA polymerase, and at least onedeoxynucleoside triphosphate comprising a nucleobase, a label and asugar, a cleavable oxymethylenedisulfide linker between the label andnucleobase, said sugar comprising a 3′-O capped by a group comprisingmethylenedisulfide as a cleavable protecting group, wherein saidnucleoside further comprises a detectable label covalently bound to thenucleobase of said nucleoside.

In one embodiment, the invention relates to a method of performing a DNAsynthesis reaction comprising the steps of a) providing a reactionmixture comprising a nucleic acid template with a primer hybridized tosaid template, a DNA polymerase, at least one deoxynucleosidetriphosphate comprising a cleavable oxymethylenedisulfide linker betweenthe label and nucleobase, with a 3′-O capped by a group comprisingmethylenedisulfide as a cleavable protecting group, and b) subjectingsaid reaction mixture to conditions which enable a DNA polymerasecatalyzed primer extension reaction. This permits incorporation of atleast one deoxynucleoside triphosphate (comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase, with a3′-O capped by a group comprising methylenedisulfide as a cleavableprotecting group) into the bound primer. In one embodiment, said DNApolymerase catalyzed primer extension reaction is part of a sequencingreaction (e.g. SBS). In one embodiment, said detectable label is removedfrom said nucleobase by exposure to a reducing agent. It is not intendedthat the invention is limited to one type of reducing agent. Anysuitable reducing agent capable of reducing disulfide bonds can be usedto practice the present invention. In one embodiment the reducing agentis phosphine [12], for example, triphenylphosphine, tributylphosphine,trihydroxymethyl phosphine, trihydroxypropyl phosphine, triscarboethoxy-phosphine (TCEP) [13,14]. In one embodiment, said reducingagent is TCEP. In one embodiment, said detectable label and 3′-OCH2-SS—Rgroup are removed from said nucleobase by exposure to compounds carryinga thiol group [15] so as to perform cleavage of dithio-based linkers andterminating (protecting) groups, such thiol-containing compoundsincluding (but not limited to) cysteine, cysteamine, dithio-succinicacid, dithiothreitol, 2,3-Dimercapto-1-propanesulfonic acid sodium salt,dithiobutylamine [16],meso-2,5-dimercapto-N,N,N′,N′-tetramethyladipamide, 2-mercapto-ethanesulfonate, and N,N′-dimethyl, N,N′-bis(mercaptoacetyl)-hydrazine [17].Reactions can be further catalyzed by inclusion of selenols [18]. Inaddition borohydrides, such as sodium borohydrides can also be used forthis purpose [19] (as well as ascorbic acid [20]. In addition, enzymaticmethods for cleavage of disulfide bonds are also known such as disulfideand thioreductase and can be used with compounds of the presentinvention [21].

In one embodiment, the invention relates to a method for analyzing a DNAsequence comprising the steps of a) providing a reaction mixturecomprising nucleic acid template with a primer hybridized to saidtemplate forming a primer/template hybridization complex, b) adding DNApolymerase, and a first deoxynucleoside triphosphate comprising anucleobase, a cleavable oxymethylenedisulfide linker between the labeland nucleobase, with a 3′-O capped by a group comprisingmethylenedisulfide as cleavable protecting group, c) subjecting saidreaction mixture to conditions which enable a DNA polymerase catalyzedprimer extension reaction so as to create a modified primer/templatehybridization complex, and d) detecting said first detectable label ofsaid deoxynucleoside triphosphate in said modified primer/templatehybridization complex. In one embodiment, the detecting allows one todetermine which type of analogue (A, T, G, C or U) has beenincorporated. In one embodiment, the method further comprises the stepsof e) removing said cleavable protecting group and optionally saiddetectable label from said modified primer/template hybridizationcomplex, and f) repeating steps b) to e) at least once (and typicallyrepeating these steps many times, e.g. 10-200 times). In one embodiment,the cleavable oxymethylenedisulfide-containing linker is hydrophobic andhas a log P value of greater than 0. In one embodiment, the cleavableoxymethylenedisulfide-containing linker is hydrophobic and has a log Pvalue of greater than 0.1. In one embodiment, the cleavableoxymethylenedisulfide-containing linker is hydrophobic and has a log Pvalue of greater than 1.0. In one embodiment, the method furthercomprises adding a second deoxynucleoside triphosphate is added duringrepeat of step b), wherein said second deoxynucleoside triphosphatecomprises a second detectable label, wherein said second detectiblelabel is different from said first detectible label. In one embodiment,the nucleobase of said second deoxynucleoside triphosphate is differentfrom the nucleobase of said first deoxynucleoside triphosphate In oneembodiment, a mixture of at least 4 differently labeled, 3′-Omethylenedisulfide capped deoxynucleoside triphosphate compoundsrepresenting analogs of A, G, C and T or U are used in step b). In oneembodiment, said mixture of at least 4 differently labeled, 3′-Omethylenedisulfide capped deoxynucleoside triphosphate compounds withthe structures:

are used in step b). In one embodiment, said mixture further comprisesunlabeled 3-O methylenedisulfide capped deoxynucleoside triphosphatecompounds such as those with the structures:

also used in step b). In one embodiment, step e) is performed byexposing said modified primer/template hybridization complex to areducing agent. In one embodiment, said reducing agent is TCEP. In oneembodiment, said detectable label is removed from said nucleobase byexposure to compounds carrying a thiol group so as to perform cleavageof dithio-based linkers and terminating (protecting) groups, suchthiol-containing compounds including (but not limited to) cysteine,cysteamine, dithio-succinic acid, dithiothreitol,2,3-Dimercapto-1-propanesulfonic acid sodium salt, dithiobutylamine,meso-2,5-dimercapto-N,N,N′,N′-tetramethyladipamide, 2-mercapto-ethanesulfonate, and N,N′-dimethyl, N,N′-bis(mercaptoacetyl)-hydrazine.

It is not intended that the present invention be limited to a particularsequencing platform. However, a preferred instrument is QIAGEN'sGeneReader DNA sequencing system (GR). In one embodiment, a DNA sequenceis determined by a method of sequencing by synthesis (SBS). In oneembodiment, each cycle of sequencing consists of eight steps: extension1, extension 2, wash 1, addition imaging solution, imaging, wash 2,cleave, and wash 3. Data collected during imaging cycles is processed byanalysis software yielding error rates, throughput values, and appliedphasing correction values. In another embodiment, an oxidative wash(Wash 11) is used after the cleave step (see FIG. 93A-C).

It is contemplated that the same or similar method could improve theperformance of other SBS platforms in general (i.e. anysequencing-by-synthesis methods that operate under similar conditions),as well as specific SBS platforms, such as HiSeq and miSeq platformsfrom Illumina; Roche 454; the Ion Torrent PGM and Proton platforms; andthe PacificBio platform.

It is not intended that the present invention be limited to only onetype of sequencing. In one embodiment, said deoxynucleoside triphosphate(comprising a nucleobase, a label and a sugar, a cleavableoxymethylenedisulfide linker between the label and nucleobase, saidsugar comprising a 3′-O capped by a group comprising methylenedisulfideas cleavable protecting group) may be used in pyrosequencing.

In one embodiment, the invention relates to a deoxynucleosidetriphosphate comprising a nucleobase and a sugar, said nucleobasecomprising a detectable label attached via a cleavableoxymethylenedisulfide linker, said sugar comprising a 3′-O capped by agroup comprising a methylenedisulfide group as a cleavable protectinggroup. In one embodiment, said nucleoside is in a mixture with apolymerase (or some other sequencing reagent). In one embodiment, thenucleobase of said nucleoside is non-natural. In one embodiment, thenon-natural nucleobase of said nucleoside is selected from the groupcomprising 7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine,and 2-amino adenine. In one embodiment, said group comprising amethylenedisulfide group is of the formula —CH₂—SS—R, wherein R isselected from the group comprising alkyl and substituted alkyl groups.In one embodiment, said mixture further comprises a primer. In oneembodiment, said primer is hybridized to nucleic acid template. In oneembodiment, said detectable label is a fluorescent label. In oneembodiment, said nucleic acid template is immobilized (e.g. in a well,channel or other structure, or alternatively on a bead).

In one embodiment, the invention relates to a method of preparing a3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside,comprising: a) providing a5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside, wherein said5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside comprises a nucleobaseand a sugar, and ii) a methylthiomethyl donor; and b) treating said5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside under conditions so asto create a3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside.In one embodiment, said methylthiomethyl donor is DMSO. In oneembodiment, said conditions comprise acidic conditions. In oneembodiment, said 5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleosidecomprises a protecting group on the nucleobase of said nucleoside. Inone embodiment, said3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleosideis purified with column chromatography.

In one embodiment, the invention relates to a method of preparing a3′-O—(R-substituted-dithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside,comprising: a) providing i) a3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside,and ii) R—SH, wherein R comprises alkyl or substituted alkyl; and b)treating said3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleosideunder conditions so as to create a3′-O—(R-substituted-dithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside.In one embodiment, said R—SH is ethanethiol. In one embodiment, saidconditions comprise basic conditions.

In one embodiment, the invention relates to a method of preparing a3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside, comprising: a)providing a3′-O—(R-substituted-dithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside;and b) treating said3′-O—(R-substituted-dithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxynucleosideunder conditions so as to create a3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside. In one embodiment,said conditions comprise exposing said3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside to NH₄F.

In one embodiment, the invention relates to a method of preparing atriphosphate of 3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside,comprising: a) providing a3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside; and b) treatingsaid 3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside underconditions so as to create a triphosphate of3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside. In one embodiment,said conditions comprises exposing said3′-O—(R-substituted-dithiomethyl)-2′-deoxynucleoside to (MeO)₃PO withPOCl₃ and Bu₃N. In one embodiment, said method further comprises step c)removal of said nucleobase protecting group. In one embodiment, saidprotecting group comprises a N-trifluoroacetyl-aminopropargyl protectinggroup. In one embodiment, said N-trifluoroacetyl-aminopropargylprotecting group is removed by solvolysis to produce a5′-O-(triphosphate)-3′-O—(R-substituted-dithiomethyl)-5-(aminopropargyl)-2′-deoxynucleoside.

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a compound wherein thestructure is:

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein R is selected from the group consisting of alkyl, substitutedalkyl groups, allyl, substituted allyl; B is a nucleobase; A is anattachment group; C is a cleavable site core; L₁ and L₂ are connectinggroups; and Label is a label. In one embodiment, said nucleobase is anon-natural nucleobase analog selected from the group consisting of7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine, and 2-aminoadenine. In one embodiment, said attachment group A is chemical groupselected from the group consisting of propargyl, hydroxymethyl,exocyclic amine, propargyl amine, and propargyl hydroxyl. In oneembodiment, said cleavable site core selected from the group consistingof:

wherein R₁ and R₂ are independently selected alkyl groups. In oneembodiment, wherein L₁ is selected from the group consisting of—CONH(CH₂)_(x)—, —COO(CH₂)_(x)—, —CO(CH₂)_(x)—, wherein x is 0-10, butmore preferably from 1-6. In one embodiment, wherein L₂ is selected fromthe group consisting of —NH—, —(CH₂)_(x)OCONH(CH₂)_(y)O(CH₂)_(z)NH—,—(CH₂)_(x)OCONH(CH₂)_(y)CH₂)_(y)O(CH₂)_(z)NH—, —CONH(CH₂)_(x)—,—CO(CH₂)—, wherein x, y, and z are each independently selected from is0-10, but more preferably from 1-6. In one embodiment, said label isselected from the group consisting of fluorophore dyes, energy transferdyes, mass-tags, biotin, and haptenes. In one embodiment, the compoundhas the structure:

wherein said label is a dye and wherein R is selected from the groupconsisting of alkyl, substituted alkyl groups, allyl, substituted allyl.In one embodiment, the compound has the structure:

wherein said label is a dye.

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein D is selected from the group consisting of an azide (—N₃),disulfide alkyl (—SS—R) and disulfide substituted alkyl groups, B is anucleobase, A is an attachment group, C is a cleavable site core, L₁ andL₂ are connecting groups, and Label is a label. In one embodiment, saidnucleobase is a natural nucleobase. In one embodiment, said nucleobaseis a non-natural nucleobase analog selected from the group consisting of7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine, and 2-aminoadenine. In one embodiment, said attachment group (A) is chemical groupselected from the group consisting of propargyl, hydroxymethyl,exocyclic amine, propargyl amine, and propargyl hydroxyl. In oneembodiment, said cleavable (C) site core selected from the groupconsisting of:

wherein R₁ and R₂ are independently selected alkyl groups. In oneembodiment, said cleavable site core selected from the group consistingof:

wherein R₁ and R₂ are independently selected alkyl groups. In oneembodiment, L₁ is selected from the group consisting of —CONH(CH₂)_(x)—,—CO—O(CH₂)_(x)—, —CONH—(OCH₂CH₂O)—, —CO—O(CH₂CH₂O)_(x)—, and—CO(CH₂)_(x)—, wherein x is 0-100. In some embodiments, x is 0-10, butmore preferably from 1-6. In one embodiment, L₂ is selected from thegroup consisting of

—NH—, —(CH₂)_(x)—NH—, —C(Me)₂(CH₂)NH—, —CH(Me)(CH₂)_(x)NH—,—C(Me)₂(CH₂)_(x)CO—, —CH(Me)(CH₂)_(x)CO—,—(CH₂)_(x)OCONH(CH₂)_(y)O(CH₂)_(z)NH—,—(CH₂)_(x)CONH(CH₂CH₂O)_(y)(CH₂)_(z)NH—, and —CONH(CH₂)_(x)—,—CO(CH₂)_(x)—, wherein x, y, and z are each independently selected fromis 0-10, but more preferably from 1-6. In one embodiment, L₂ is selectedfrom the group consisting of —NH—, —(CH₂)_(x)—NH—, —C(Me)₂(CH₂)_(x)NH—,—CH(Me)(CH₂)_(x)NH—, —C(Me)₂(CH₂)_(x)CO—, —CH(Me)(CH₂)_(x)CO—,—(CH₂)_(x)OCONH(CH₂)_(y)O(CH₂)_(z)NH—, and —CONH(CH₂)_(x)—,—CO(CH₂)_(x)—, wherein x, y, and z are each independently selected fromis 0-100. In one embodiment, x, y, and z are each independently selectedfrom is 0-10, but more preferably from 1-6. In one embodiment, saidlabel is selected from the group consisting of fluorophore dyes, energytransfer dyes, mass-tags, biotin, and haptenes. In one embodiment, thecompound has the following structure (while a particular nucleobase andlabel are shown below, other analogous nucleotide counterparts arecontemplated, i.e. any of the various labels in the specification andfigures could be substituted, and the nucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

embodiment, the compound has the following structure (while a particularnucleobase and label are shown below, other analogous nucleotidecounterparts are contemplated, i.e. any of the various labels in thespecification and figures could be substituted, and the nucleobase couldbe different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the compound has the following structure (while aparticular nucleobase and label are shown below, other analogousnucleotide counterparts are contemplated, i.e. any of the various labelsin the specification and figures could be substituted, and thenucleobase could be different):

In one embodiment, the present invention contemplates unlabeledcompounds. In one embodiment, the compound has the structure:

wherein R is selected from the group consisting of alkyl, substitutedalkyl groups, allyl, substituted allyl; and B is a nucleobase. In oneembodiment, the compound has the structure (again B is a nucleobase):

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, the compound has the structure:

In one embodiment, said nucleobase is a non-natural nucleobase analogselected from the group consisting of 7-deaza guanine, 7-deaza adenine,2-amino,7-deaza adenine, and 2-amino adenine.

In one embodiment, the invention relates to a method of synthesizing3′-OCH2-SSMe nucleotide analogs using3′-(2,4,6-trimethoxyphenyl)methanethiol nucleoside as intermediate, andDMTSF and dimethyldisulfide as sulfur source shown in FIG. 43.

In one embodiment, the invention relates to a labeled deoxynucleosidetriphosphate according to the following structure:

wherein D is selected from the group consisting of an azide, disulfidealkyl, disulfide substituted alkyl groups, disulfide allyl, anddisulfide substituted allyl groups; B is a nucleobase; Linker comprisesa cleavable oxymethylenedisulfide-containing site core. In oneembodiment, said cleavable site core is selected from the groupconsisting of:

wherein R₁ and R₂ are independently selected alkyl groups; and Label isa label. In one embodiment, said Linker is hydrophobic. In oneembodiment, said Linker has a log P value of greater than 0. In oneembodiment, said Linker has a log P value of greater than 0.1. In oneembodiment, said Linker has a log P value of greater than 0.5. In oneembodiment, said Linker has a log P value of greater than 1.0.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, “hydrogen” means —H; “hydroxy” means —OH; “oxo” means═O; “halo” means independently —F, —Cl, —Br or —I; “amino” means —NH₂(see below for definitions of groups containing the term amino, e.g.,alkylamino); “hydroxyamino” means —NHOH; “nitro” means —NO₂; “imino”means ═NH (see below for definitions of groups containing the termimino, e.g., alkylamino); “cyano” means —CN; “azido” means —N₃;“mercapto” means —SH; “thio” means ═S; “sulfonamido” means —NHS(O)₂—(see below for definitions of groups containing the term sulfonamido,e.g., alkylsulfonamido); “sulfonyl” means —S(O)₂— (see below fordefinitions of groups containing the term sulfonyl, e.g.,alkylsulfonyl); and “silyl” means —SiH₃ (see below for definitions ofgroup(s) containing the term silyl, e.g., alkylsilyl).

As used herein, “methylene” means a chemical species in which a carbonatom is bonded to two hydrogen atoms. The —CH₂— group is considered tobe the standard methylene group. Methylene groups in a chain or ringcontribute to its size and lipophilicity. In this context dideoxy alsorefers the methylene groups. In particular a 2,3-dideoxy compound is thesame as 2,3-methylene (2,3-methylene-glycoside=2,3-dideoxy-glycoside).

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group; “(C≤n)” defines the maximum number (n) ofcarbon atoms that can be in the group; (C_(n-n′)) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group. Forexample, “alkoxy_((C≤10))” designates those alkoxy groups having from 1to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)). Similarly,“alkyl_((C2-10))” designates those alkyl groups having from 2 to 10carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)).

The term “alkyl” when used without the “substituted” modifier refers toa non-aromatic monovalent group with a saturated carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr or i-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl or sec-Bu), —CH₂CH(CH₃)₂(iso-butyl or i-Bu), —C(CH₃)₃ (tert-butyl or t-Bu), —CH₂C(CH₃)₃(neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “substituted alkyl”refers to a non-aromatic monovalent group with a saturated carbon atomas the point of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and at leastone atom independently selected from the group consisting of N, O, F,Cl, Br, I, Si, P, and S. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂C, —CH₂Br, —CH₂SH, —CF₃,—CH₂CN, —CH₂C(O)H, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)NHCH₃,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, —CH₂CF₃, —CH₂CH₂OC(O)CH₃,—CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The terms “cleavable oxymethylenedisulfide linker” and “cleavableoxymethylenedisulfide-containing linker” are meant to indicate that thelinker comprises an oxymethylenedisulfide group, and are not to beconsidered limited to only an oxymethylenedisulfide group, but ratherlinkers that may contain more than just that group, for example as seenin the compounds in FIG. 25. Similarly, the terms “oxymethylenedisulfidesite core” and “oxymethylenedisulfide-containing site core” are meant toindicate that the site core comprises an oxymethylenedisulfide group,and are not to be considered limited to only an oxymethylenedisulfidegroup, but rather site cores that may contain more than just that group.

The term “nucleic acid” generally refers to both DNA or RNA, whether itis a product of amplification, synthetically created, products ofreverse transcription of RNA or naturally occurring. Typically, nucleicacids are single- or double-stranded molecules and are composed ofnaturally occurring nucleotides. Double-stranded nucleic acid moleculescan have 3′- or 5′-overhangs and as such are not required or assumed tobe completely double-stranded over their entire length. Furthermore, thenucleic acid can be composed of non-naturally occurring nucleotidesand/or modifications to naturally occurring nucleotides. Examples arelisted herein, but are not limited to: phosphorylation of 5′ or 3′nucleotides to allow for ligation or prevention of exonucleasedegradation/polymerase extension, respectively; amino, thiol, alkyne, orbiotinyl modifications for covalent and near covalent attachments;fluorophores and quenchers; phosphorothioate, methylphosphonates,phosphoroamidates and phosphotriester linkages between nucleotides toprevent degradation; methylation; and modified bases or nucleosides suchas deoxy-inosine, 5-bromo-dU, 2′-deoxy-uridine, 2-aminopurine,2′,3′-dideoxy-cytidine, 5-methyl-dC, locked nucleic acids (LNA's),iso-dC and -dG bases, 2′-O-methyl RNA bases and fluorine modifiednucleosides.

In some of the methods contemplated herein, primers are at leastpartially complementary to at least a portion of template to besequenced. The term “complementary” generally refers to the ability toform favorable thermodynamic stability and specific pairing between thebases of two nucleotides (e.g. A with T) at an appropriate temperatureand ionic buffer conditions. This pairing is dependent on the hydrogenbonding properties of each nucleotide. The most fundamental examples ofthis are the hydrogen bond pairs between thymine/adenine andcytosine/guanine bases. In the present invention, primers foramplification of target nucleic acids can be both fully complementaryover their entire length with a target nucleic acid molecule or“semi-complementary” wherein the primer contains an additional,non-complementary sequence minimally capable or incapable ofhybridization to the target nucleic acid.

The term “hybridize” generally refers to the base-pairing betweendifferent nucleic acid molecules consistent with their nucleotidesequences. The terms “hybridize” and “anneal” can be usedinterchangeably.

The term “oligonucleotide” generally refers to a nucleic acid sequencetypically designed to be single-stranded DNA and less than 75nucleotides in length.

The term “primer” generally refers to an oligonucleotide that is able toanneal, or hybridize, to a nucleic acid sequence and allow for extensionunder sufficient conditions (buffer, dNTP's, polymerase, mono- anddivalent salts, temperature, etc. . . . ) of the nucleic acid to whichthe primer is complementary.

The terms “template nucleic acid”, “template molecule”, “target nucleicacid”, and “target molecule” can be used interchangeably and refer to anucleic acid molecule that is the subject of an amplification reactionthat may optionally be interrogated by a sequencing reaction in order toderive its sequence information. The template nucleic acid may be anucleic acid which has been generated by a clonal amplification methodand which may be immobilized on a solid surface, i.e. immobilized onbeads or an array.

The term “nucleoside” refers to a compound consisting of a base linkedto the C-1′ carbon of a sugar, for example, ribose or deoxyribose. Thebase portion of the nucleoside is usually a heterocyclic base, e.g., apurine or pyrimidine.

The term “nucleotide” refers to a phosphate ester of a nucleoside, as amonomer unit or within a polynucleotide. “Nucleoside 5′-triphosphate”refers to a nucleotide with a triphosphate ester group attached to thesugar 5′-carbon position, and is sometimes denoted as “NTP”, “dNTP”(2′-deoxynucleoside triphosphate or deoxynucleoside triphosphate) and“ddNTP” (2′,3′-dideoxynucleoside triphosphate or dideoxynucleosidetriphosphate). “Nucleoside 5′-tetraphosphate” refers to an alternativeactivated nucleotide with a tetraphosphate ester group attached to thesugar 5′-carbon position. PA-nucleotide refers to a propargyl analogue.

The term “protecting group,” as that term is used in the specificationand/or claims, is used in the conventional chemical sense as a group,which reversibly renders unreactive a functional group under certainconditions of a desired reaction and is understood not to be H. Afterthe desired reaction, protecting groups may be removed to deprotect theprotected functional group. In a preferred embodiment, all protectinggroups should be removable (and hence, labile) under conditions which donot degrade a substantial proportion of the molecules being synthesized.A protecting group may also be referred to as a “capping group” or a“blocking group” or a “cleavable protecting group.” It should be notedthat, for convenience, the functionality protected by the protectinggroup may also be shown or referred to as part of the protecting group.In the context of the nucleotide derivatives described herein, aprotecting group is used on the 3′ position. It is not intended that thepresent invention be limited by the nature or chemistry of thisprotecting group on the reversibly terminating nucleotides used insequencing. A variety of protecting groups is contemplated for thispurpose, including but not limited to: 3′-O-azidomethyl nucleotides,3′-O-aminoxy nucleotides, 3′-O-allyl nucleotides; and disulfidenucleotides, 3′-O-azidoalkyl, 3′-O-dithiomethyl alkyl, 3′-O-dithiomethylaryl, 3′-O-acetyl, 3′-O-carbazate, 3′-O-alkyl ether, 3′-O-alkyl ester,3′-O-aldoxime (—O—N═CH—R), 3′-O-ketoxime (—O—N═C(R, R′)).

One embodiment of the present invention contemplates attaching markersdirectly on the 3′-OH function of the nucleotide via functionalizationof the protective groups.

The term “label” or “detectable label” in its broadest sense refers toany moiety or property that is detectable, or allows the detection ofthat which is associated with it. For example, a nucleotide, oligo- orpolynucleotide that comprises a label is detectable. Ideally, a labeledoligo- or polynucleotide permits the detection of a hybridizationcomplex, particularly after a labeled nucleotide has been incorporatedby enzymatic means into said hybridization complex of a primer and atemplate nucleic acid. A label may be attached covalently ornon-covalently to a nucleotide, oligo- or polynucleotide. In variousaspects, a label can, alternatively or in combination: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the second label, e.g., FRET; (iii)stabilize hybridization, e.g., duplex formation; (iv) confer a capturefunction, e.g., hydrophobic affinity, antibody/antigen, ioniccomplexation, or (v) change a physical property, such as electrophoreticmobility, hydrophobicity, hydrophilicity, solubility, or chromatographicbehavior. Labels vary widely in their structures and their mechanisms ofaction. Examples of labels include, but are not limited to, fluorescentlabels, non-fluorescent labels, colorimetric labels, chemiluminescentlabels, bioluminescent labels, radioactive labels, mass-modifyinggroups, antibodies, antigens, biotin, haptens, enzymes (including, e.g.,peroxidase, phosphatase, etc.), and the like. To further illustrate,fluorescent labels may include dyes of the fluorescein family, dyes ofthe rhodamine family, dyes of the cyanine family, or a coumarine, anoxazine, a boradiazaindacene or any derivative thereof. Dyes of thefluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE. Dyesof the rhodamine family include, e.g., Texas Red, ROX, R110, R6G, andTAMRA. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, and TAMRA arecommercially available from, e.g., Perkin-Elmer, Inc. (Wellesley, Mass.,USA), Texas Red is commercially available from, e.g., Life Technologies(Molecular Probes, Inc.) (Grand Island, N.Y.). Dyes of the cyaninefamily include, e.g., CY2, CY3, CY5, CY5.5 and CY7, and are commerciallyavailable from, e.g., GE Healthcare Life Sciences (Piscataway, N.J.,USA).

The term “differently labeled,” as used herein, refers to the detectiblelabel being a different label, rather than the label being found in adifferent position upon the labeled nucleoside nucleobase.

The term “analogs of A, G, C and T or U” refers to modifieddeoxynucleoside triphosphate compounds, wherein the nucleobase of saiddeoxynucleoside closely resembles the corresponding nucleosideDeoxyadenosine, Deoxyguanosine, Deoxycytidine, and Thymidine orDeoxyuridine.

In the case of detectable labeled deoxynucleoside triphosphate compoundsan analog of A or Deoxyadenosine would be represented as

although it is preferred that there be a linker between the nucleobaseand the label. In the case of detectable labeled deoxynucleosidetriphosphate compounds an analog of G or Deoxyguanosine would berepresented as

although it is preferred that there be a linker between the nucleobaseand the label. In the case of detectable labeled deoxynucleosidetriphosphate compounds an analog of C or Deoxycytidine would berepresented as

although it is preferred that there be a linker between the nucleobaseand the label. In the case of detectable labeled deoxynucleosidetriphosphate compounds an analog of T or U or Thymidine or Deoxyuridinewould be represented as

although it is preferred that there be a linker between the nucleobaseand the label. Additional nucleobase may include: non-natural nucleobaseselected from the group consisting of 7-deaza guanine, 7-deaza adenine,2-amino,7-deaza adenine, and 2-amino adenine. In the case of analogs,the detectable label may also include a linker section between thenucleobase and said detectable label.

The term “TCEP” or “tris(2-carboxyethyl)phosphine)” refers to a reducingagent frequently used in biochemistry and molecular biologyapplications. It is often prepared and used as a hydrochloride salt(TCEP-HCl) with a molecular weight of 286.65 gram/mol. It is soluble inwater and available as a stabilized solution at neutral pH andimmobilized onto an agarose support to facilitate removal of thereducing agent. It is not intended that the invention is limited to onetype of reducing agent. Any suitable reducing agent capable of reducingdisulfide bonds can be used to practice the present invention. In oneembodiment the reducing agent is phosphine [12], for example,triphenylphosphine, tributylphosphine, trihydroxymethyl phosphine,trihydroxypropyl phosphine, tris carboethoxy-phosphine (TCEP) [13, 14].It is not intended that the present invention be limited to the use ofTCEP. In one embodiment, said detectable label and 3′-OCH2-SS—R groupare removed from said nucleobase by exposure to compounds carrying athiol group so as to perform cleavage of dithio-based linkers andterminating (protecting) groups, such thiol-containing compoundsincluding (but not limited to) cysteine, cysteamine, dithio-succinicacid, dithiothreitol, 2,3-Dimercapto-1-propanesulfonic acid sodium salt,dithiobutylamine, meso-2,5-dimercapto-N,N,N′,N′-tetramethyladipamide,2-mercapto-ethane sulfonate, and N,N′-dimethyl,N,N′-bis(mercaptoacetyl)-hydrazine [17]. Reactions can be furthercatalyzed by inclusion of selenols [18]. In addition borohydrides, suchas sodium borohydrides can also be used for this purpose [19](as well asascorbic acid [20]. In addition, enzymatic methods for cleavage ofdisulfide bonds are also well known such as disulfide and thioreductaseand can be used with compounds of the present invention [21].

N-Cyclohexyl-2-aminoethanesulfonic acid, also known as CHES, is abuffering agent.

High pH is defined as a pH in the range of 9.0-10.0, e.g. pH 9.5. Low pHis defined as 8.5 and lower.

ATP on FFPE sample type is indicated as ATPf (“f” for FFPE)

ATP on liquid biopsy sample type is indicated as ATPp (“p” for plasma)

“Acrometrix” refers to the commercially available DNA sample type used.

Reactions can take place in a variety of places, e.g. wells, channels,slides, flow cells, etc. Flow cells can have channels, as described inU.S. Pat. No. 8,940,481, hereby incorporated by reference. Flow cellscan be on moving supports within instruments as described in U.S. Pat.No. 8,900,810, hereby incorporated by reference.

In a preferred embodiment, the present invention contemplates the use ofa sequencing additive, namely CMIT, in an extend reagent (e.g. abuffered solution of nucleotide analogues and polymerase) for the primerextension step. CMIT is commercially available. For example, the reagent“Proclin300” (which is a mixture of5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT);2-Methyl-4-isothiazolin-3-one (MIT); glycol: 93-95%; modified alkylcarboxylate: 2-3%) is commercially available from Sigma-Aldrich.Structures for these components are shown in FIG. 95. A small amount ofProclin300 can be added to the buffered solution, e.g. 15 ul ofProclin300 can be added to 15 ml of extend reagent, and be effective.While not intending in any manner to limit the present invention to anyparticular mechanism of action, it is believed that the CMIT in“Proclin300” inhibits (and may even prevent) terminator cleavage duringthe extension reaction, and in particular, during sequencing reactionswhere deoxynucleoside triphosphates comprising a 3′-O position capped bya disulfide-based 3′-terminator group are used. In a preferredembodiment, Proclin300 is added fresh to the extend reagent. While itmay be used when it is under 48 hours old (and more preferably less than24 hours old), it is most preferred that the extend reagent withProclin300 is made just before (e.g. within minutes to 2 hours before)the sequencing run. It is also preferred that the oxidative scavengerH₂O₂ not be used when CMIT is used as an additive. When CMIT is used,the preferred scavenger is cystamine.

Regardless of the mechanism, the use of CMIT (e.g. in Proclin300) in theextension step is observed to control (and even reduce) “lead.” Lead asa function per cycle is one of the key metric of sustaining sequencingperformance. When lead is observed to be a linear function increasingfrom 0.0 to 0.5, it is deemed to be “within spec.”

Regardless of the mechanism, the use of CMIT (e.g. in Proclin300) in theextension step is observed to increase read length. This could not bepredicted.

Regardless of the mechanism, the use of CMIT in the extension step isobserved to decrease DNA template damage. DNA template damage has beenobserved to be more prominent at the outlet region of the flow cell dueto fluidic patterns and manifests itself as a read quality degradationgradient from the inlet to the outlet region of the flow cell. With CMITin the extend reagent, a dramatic improvement in inlet-to-outlet isobserved.

In some embodiments, the method of sequencing includes a pre-Extend washstep prior to the extension step. Without being bound to any mechanismor theory, it is believed that the pre-Extend wash step is a mitigationmeasure to ensure that any potential cystamine/cysteamine by-products(e.g. in the post-cleave step from a previous cycle) do not carry overinto the Extension step. In a preferred embodiment, the pre-Extend washdoes not contain methylenediphosphonic acid (PcPi). It is preferred thatmethylenediphosphonic acid is used in an earlier wash step to preventnucleotide build up on the surface (e.g. in the Wash 9/10 step).

DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part ofthe specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The figures are only for the purpose ofillustrating a preferred embodiment of the invention and are not to beconstrued as limiting the invention.

FIG. 1 shows examples of nucleoside triphosphates with 3′-O capped by agroup comprising methylenedisulfide, where the R represents alkyl groupsuch as methyl, ethyl, isopropyl, t-butyl, n-butyl, or their analogswith substituent group containing hetero-atoms such as O, N, S etc.

FIG. 2 shows labeled analogs of nucleoside triphosphates with 3′-Omethylenedisulfide-containing protecting group, where labels areattached to the nucleobase via cleavable oxymethylenedisulfide linker(—OCH₂—SS—). The analogs are (clockwise from the top left) forDeoxyadenosine, Thymidine or Deoxyuridine, Deoxycytidine andDeoxyguanosine.

FIG. 3 shows a step-wise mechanism of deprotection of the 3′-Oprotection group with a reducing agent, such as TCEP.

FIG. 4 shows the cleavage reactions products a traditional sulfide andoxymethylene sulfide linked labeled nucleotides.

FIG. 5 shows an example of the labeled nucleotides where the spacer ofthe cleavable linker includes the propargyl ether linker. The analogsare (clockwise from the top left) for Deoxyadenosine, Thymidine orDeoxyuridine, Deoxycytidine and Deoxyguanosine.

FIG. 6 shows an example of the labeled nucleotides where the spacer ofthe cleavable linker includes the propargylamine linker. The analogs are(clockwise from the top left) for Deoxyadenosine, Thymidine orDeoxyuridine, Deoxycytidine and Deoxyguanosine.

FIG. 7 shows an example of the labeled nucleotides where the spacer ofthe cleavable linker includes the methylene (—(CH₂)_(n)— directlyattached to the nucleobases at 5-position for pyrimidine, and at7-de-aza-carbon for purines. This linker may be methylene (n=1) orpolymethylene (n>1) where after cleavage, the linker generates—(CH₂)_(n)OH group at the point of attachment on the nucleobases, andwhere the L₁ and L₂ represent spacers, and substituents R₁, R₂, R3 andR4 are group of atoms that provide stability to the cleavable linker asdescribed earlier. The analogs are (clockwise from the top left) forDeoxyadenosine, Thymidine or Deoxyuridine, Deoxycytidine andDeoxyguanosine.

FIG. 8 shows a synthesis of the unlabeled dT analog (compound 5).

FIG. 9 shows the synthesis of 3′-O-(ethyldithiomethyl)-dCTP (10).

FIG. 10 shows a synthetic route of the labeled nucleotides specific forlabeled dT intermediate.

FIG. 11 shows a cleavable linker synthesis starting from an1,4-dutanediol.

FIG. 12 shows another variant of cleavable linker, where the stabilizinggem-dimethyl group is attached to α-carbon of the cleavable linker.

FIG. 13 shows the synthesis of a cleavable linker, where the disulfideis flanked by gem-dimethyl groups and attached to a flexible ethyleneglycol linker (PEG). The linker is attached to the PA-nucleotide viacarbamate group (—NH—C(═O)O—). The resulting nucleotide analogue in suchcase can be as in compound 35 (dUTP analogue).

FIG. 14 shows the synthesis of a cleavable linker for dATP analoguewhere the cleavable disulfide is flanked by gem-dimethyl group and thelinker is attached to PA-nucleotide via urea group (—NH(C═O)NH—). Forother nucleotide analogues (e.g. for analogues of dCTP, dGTP, dUTP) canbe synthesized similarly replacing 42 by appropriate PA-analogues at thelast step of the reaction sequence.

FIG. 15 shows the synthesis of a cleavable linker compound 45, where thelinker is tethered to PA-nucleotides via urea functionality and thedisulfide is connected to the dye by a two carbon linker. The resultingnucleotide analogue in such case can be as in compound 49 (dGTPanalogue). Other nucleotide analogues (e.g. analogues of dATP, dUTP,dCTP) can be synthesized similarly by replacing nucleotide 46 withappropriate PA-nucleotide analogues in the third step of the reactionsequence.

FIG. 16 shows that when labeled nucleotide 50 was exposed to 10 eq ofTCEP at 65° C., it generated a number of side products includingcompound 52 along with the expected product 51.

FIG. 17 shows an LC-MS trace of the TCEP exposed product of compound 50,extracted at 292 nm (bottom) and 524 nm (top), analyzed after 5 minutesexposure, where peak at 11.08 min corresponds to compound 51, peak at10.88 min to compound 52 and other peaks to side products.

FIG. 18 shows an LC-MS trace of the TCEP exposed product of compound 50,extracted at 292 nm (bottom) and 524 nm (top), analyzed after 15 minutesexposure; where peak at 11.32 min corresponds to compound 51 and otherpeaks to side products.

FIG. 19 shows that under identical cleavage conditions, theoxymethylenedisulfide linked nucleotide 35 cleanly produced the desiredcleavage products, compounds 53 and 54. The methylene thiol segment(—CH₂SH) of the linker was fully eliminated from the nucleotide uponcleavage of the disulfide group FIG. 20 shows an LC-MS trace of the TCEPexposed product of compound 35, extracted at 292 nm (bottom) and 524 nm(top), analyzed after 5 minutes exposure, where peak at 11.24 mincorresponds to compound 53 and peak at 34.70 min to compound 54.

FIG. 21 shows LC-MS trace of the TCEP exposed product of compound 35,extracted at 292 nm (bottom) and 524 nm (top), analyzed after 15 minutesexposure, where peak at 11.25 min corresponds to compound 53 and peak at34.70 min to compound 54.

FIG. 22 shows the synthesis of 3′-OCH₂—SS-Me analogues with thereplacement of mercaptoethanol (EtSH) by methanethiol or sodiumthiomethoxide at the appropriate step, different from that of3′-OCH2-SS-Et (FIG. 10).

FIG. 23 shows the coupling of PA-nucleotide (e.g. 57) to the appropriatecleavable —OCH₂—SS— linkers, and finally to fluorophore dye using theactivated linker 32.

FIG. 24 shows nucleotide analogues with different linker achieved,compounds 60 and 61.

FIG. 25 shows the structure of 4-nucleotide analogues labeled bydifferent fluorophore reporting groups, where R=Me- or Et-.

FIG. 26 shows the structure of 4-nucleotide analogues labeled bydifferent fluorophore reporting groups, where R=Me- or Et-group.

FIG. 27 shows the structure of 4-nucleotide analogues labeled bydifferent fluorophore reporting groups, where R=Me- or Et-group.

FIG. 28 shows Generic universal building blocks structures comprisingnew cleavable linkers of present invention. PG=Protective Group, L1,L2-linkers (aliphatic, aromatic, mixed polarity straight chain orbranched). RG=Reactive Group. In one embodiment of present inventionsuch building blocks carry an Fmoc protective group on one end of thelinker and reactive NHS carbonate or carbamate on the other end. Thispreferred combination is particularly useful in modified nucleotidessynthesis comprising new cleavable linkers. A protective group should beremovable under conditions compatible with nucleic acid/nucleotideschemistry and the reactive group should be selective. After reaction ofthe active NHS group on the linker with amine terminating nucleotide, anFmoc group can be easily removed using base such as piperidine orammonia, therefore exposing amine group at the terminal end of thelinker for the attachment of cleavable marker. A library of compoundscomprising variety of markers can be constructed this way very quickly.

FIG. 29 shows generic structure of nucleotides carrying cleavable markerattached via novel linker of present invention. S=sugar (i.e., ribose,deoxyribose), B=nucleobase, R═H or reversibly terminating group(protective group). Preferred reversibly terminating groups include butare not limited to: Azidomethyl (—CH₂N₃), Dithio-alkyl (—CH₂—SS—R),aminoxy (—ONH₂).

FIG. 30 shows another generic structure for nucleotides carryingcleavable marker attached via the cleavable linker of present invention,wherein D is selected from the group comprising an azide, disulfidealkyl and disulfide substituted alkyl groups, B is a nucleobase, A is anattachment group, C is a cleavable site core, L₁ and L₂ are connectinggroups, and Label is a label (in the compounds with a label).

FIG. 31 shows the chemical structures of compounds (L-series (96),B-series (97), (A-series, (98), and (G-series (99) family) tested inFIG. 32A-C.

FIG. 32A shows a time course of incorporation of 3′-O-azidomethylAlexa488 labeled nucleotide analogs with various disulfide basedcleavable linkers: L-Series (96), B-series (97), A-series (98), andG-series (99) family.

FIG. 32B shows reaction rates of incorporation for 3′-O-azidomethylAlexa488 labeled nucleotide analogs with various disulfide basedcleavable linkers: L-series (96), B-series (97), A-series (98), andG-series (99) family.

FIG. 32C shows reaction rates of incorporation for 3′-O-azidomethylAlexa488 labeled nucleotide analogs with various disulfide basedcleavable linkers: L-series (96), B-series (97), A-series (98), andG-series (99) family vs concentration of nucleotides.

FIG. 33 shows incorporation kinetics for the dA 3′-reversiblyterminating nucleotides: —CH₂—N₃, —CH₂—SS-Et, —CH₂—SS-Me.

FIG. 34 shows incorporation kinetics of dC 3′-reversibly terminatingnucleotide with 3′-O—CH2-SS-Et terminating group with 3 different DNApolymerases: T9, J5 and J8.

FIG. 35 shows optimized concentrations of nucleotides used in Extend Areactions on GR sequencer [nM].

FIG. 36 shows sequencing performance of A-series (98) nucleotides asmeasured by raw error rate.

FIG. 37 shows sequencing performance of A-series (98) nucleotides asmeasured by percentage of perfect (error free) reads.

FIG. 38 shows sequencing performance of A-series (98) nucleotides asmeasured by variety of sequencing metrics.

FIG. 39 shows sequencing performance of G-series (99) nucleotides asmeasured by raw error rate.

FIG. 40 shows sequencing performance of G-series (99) nucleotides asmeasured by percentage of perfect (error free) reads.

FIG. 41 shows identification of multiplex barcodes from sequencing runscontaining 3′-O—CH₂—SS-Et nucleotides in ExtB and in both ExtB and A.

FIG. 42 shows a comparison of stability at elevated temperature inExtend A buffer of labeled, reversibly terminating dC with variouscleavable linkers: B═B-series (97, 116, 117, and 118), G=G-series (99,103, 104, and 105), A=A-series (98, 100, 101, and 102), and SS=L-series(96, 50, 106, and 115).

FIG. 43 shows a synthetic scheme illustrating the synthesis of compounds63-67 from compound 62. The synthesis is described in Example 33,Example 34 and Example 35.

FIG. 44 shows a synthetic scheme illustrating the synthesis of compounds69-71 and 119-120 from compound 68. The synthesis is described inExamples 36-38.

FIG. 45 shows complete chemical structures of four labeled nucleotidescorresponding to dCTP, dTTP, dATP and dGTP from top to bottom (A-series,98, 100, 101, and 102).

FIG. 46 shows complete chemical structures of four labeled nucleotidescorresponding to dCTP, dTTP, dATP and dGTP from top to bottom (G-series,99, 103, 104, and 105).

FIG. 47 shows complete chemical structures of four labeled nucleotidescorresponding to dCTP, dTTP, dATP and dGTP from top to bottom (L-series,96, 50, 106, and 115).

FIG. 48 shows complete chemical structures of four labeled nucleotidescorresponding to dCTP, dTTP, dATP and dGTP from top to bottom (B-series:compounds 97, 116, 117, and 118).

FIG. 49 shows example concentrations of nucleotides used in sequencingon GR instrument (labeled, compounds 72, 74, 76, 78) and non-labeled(compounds 120, 126, 132, 138), all carrying the —CH₂—SS-Me on their 3′as reversibly terminating group.

FIG. 50 shows example of intensities generated in sequencing run on GRusing novel nucleotides (labeled and non-labeled as in described forFIG. 49), all carrying the —CH₂—SS-Me).

FIG. 51 shows a series of non-linker examples of nucleosidetriphosphates with 3′-O capped by a group comprising methylenedisulfidemethyl.

FIG. 52 shows the structure of 4-nucleotide analogues labeled bydifferent fluorophore reporting groups with 3′-O capped by a groupcomprising methylenedisulfide methyl.

FIG. 53 is a schematic that shows one embodiment of a synthesis of NHSactivated form of common linker.

FIG. 54 is a schematic that shows one embodiment of the synthesis ofMeSSdATP.

FIG. 55 is a schematic that shows one embodiment of the synthesis ofMeSSdCTP.

FIG. 56 is a schematic that shows one embodiment of the synthesis ofMeSSdGTP.

FIG. 57 is a schematic that shows one embodiment of the synthesis ofMeSSdTTP.

FIG. 58 is a schematic that shows one embodiment of the synthesis ofMeSSdATP-PA.

FIG. 59 is a schematic that shows one embodiment of the synthesis of 76.

FIG. 60 is a schematic that shows one embodiment of the synthesis ofMeSSdCTP-PA.

FIG. 61 is a schematic that shows one embodiment of the synthesis of 72.

FIG. 62 is a schematic that shows one embodiment of the synthesis ofMeSSdGTP-PA.

FIG. 63 is a schematic that shows one embodiment of the synthesis ofMeSSdGTP-ARA-Cy5.

FIG. 64 is a schematic that shows one embodiment of the synthesis ofMeSSdUTP-PA.

FIG. 65 is a schematic that shows one embodiment of the synthesis of 74.

FIG. 66 is a schematic that shows the structures of3′-OCH₂S-(2,4,6-trimethoxyphenyl)methane-dNTPs.

FIG. 67 is a schematic that shows one embodiment of the synthesis of3′-(OCH₂SSMe)-dNTPs from 3′-OCH₂S-(2,4,6-trimethoxyphenyl)methane-dNTPs.

FIG. 68 shows the key intermediates for the synthesis of3′-(OCH₂SSMe)-dNTP-PAs.

FIG. 69 is a schematic that shows one embodiment of the synthesis of3′-(OCH₂SSMe)-dNTP-PA from3′-OCH₂S-(2,4,6-trimethoxyphenyl)methane-dNTP-PAs.

FIG. 70 is a schematic that shows linker installation and conjugation offluorescent dye.

FIG. 71 shows structures of hydroxymethyl derivatives nucleobasesderivatives that could be used to attach linkers and terminating groupsof the present invention. R=reversibly terminating group, CL=cleavablelinker of the present invention.

FIG. 72 shows structures of hydroxymethyl derivatives nucleobasesderivatives after cleavage has been performed.

FIG. 73A-I shows examples of compounds carrying thiol, function thatcould be used to perform cleavage of dithio-based linkers andterminating groups of the present invention: 73A)—cysteamine,73B)—dithio-succinic acid, 73C)—cysteamine, 73D)—dithiothreitol,73E)—2,3-Dimercapto-1-propanesulfonic acid sodium salt,73F)—dithiobutylamine,73G)—meso-2,5-dimercapto-N,N,N′,N′-tetramethyladipamide, 73H)2-mercaptoethane sulfonate, 73I)—N,N′-dimethyl,N,N′-bis(mercaptoacetyl)-hydrazine.

FIG. 74A-C shows an example of selective and stepwise cleavage of linkerand 3′-protective group—chemical structures and reaction scheme.

FIG. 75A-C shows an example of selective and stepwise cleavage oflinker-chromatograms associated with each step of the cleavage.

FIG. 76 shows an example of selective and stepwise cleavage oflinker—absorption spectar extracted from peaks corresponding to allsteps of selective cleavage reactions.

FIG. 77 shows cleavage reaction scheme for nucleotide bearing dithioprotecting group on te 3′ and ditho based linker.

FIG. 78A shows chromatograms of starting material and cleavage reactionmixtures analyzed by RP-HPLC after 10 minutes of incubation with cleavereagents: dithiosuccinic acid, L-cysteine, dithiothreitol (DTT) andcysteamine.

FIG. 78B shows compositions of reaction mixtures as analyzed by RP-HPLC.

FIG. 79 presents exemplary dithiol compounds at a vicinal postion orseparated by at least one hydrocarbon and alternatively attached to ahydrocarbon spacer with an anionic functional group (R).

FIG. 80 presents exemplary a thiol compound comprising a functionalgroup at an adjacent hydrocarbon.

FIG. 81 presents an exemplary mechanism of disulfide bond reduction by2,3-dimercaptopropanedulfonate(DMPS).

FIG. 82 presents one embodiment of the present invention showingcleavage of 3′-(MeSSCH2) capped nucleotide with attached disulfidelinker by thiol based cleave reagents (the LC-MS data for Compounds A, Band C is shown in FIG. 84).

FIG. 83 presents representative structures of various thiol based cleavereagents using in the cleavage study presented in Table 1.

FIG. 84 presents exemplary LC-MS data of the cleavage studies ofcompound A; 15.8 min compound A, 14.0 min compound B, and 7.10 mincompound C.

FIG. 85 presents one embodiment to synthesize3′-O-(methylmethylenedisulfide) 5-(N-triflouroacetyl-aminopropargyl)-dU((MeSSdU-PA(COCF3), 7) in accordance with Example 84.

FIG. 86 presents one embodiment to synthesize3′-O-(methylinethylenedisulfide)-5-(aminopropargyl)-2′-deoxyuridine(MeSSdUTPPA,8) in accordance with Example 85.

FIG. 87A-C presents a schematic of one embodiment of a kit comprisingreagents as disclosed herein to perform SBS sequencing.

FIG. 87A: An exemplary Box IA comprising containers of extension premixand a cleave base buffer.

FIG. 87B: An exemplary Box 1B comprising containers of base wash premixand wash buffer.

FIG. 87C: An exemplary Box 2 comprising containers of extension add-onreagents, a DNA polymerase and an imaging reagent.

FIG. 87D: An exemplary Box 3 comprising containers of wash add-onreagents and a cleave-solid add-on reagent.

FIG. 88 presents exemplary data showing a dose response of DMPS CHEShigh pH cleave reagent on the raw error rate of an SBS sequencingmethod.

FIG. 89A-C presents exemplary SBS sequencing data showing a lead/lagcomparision using a DMPS TRIS high pH cleave reagent and a DMPS CHESreagent.

FIG. 89A; high pH DMPS TRIS cleave reagent.

FIG. 89B: DMPS CHES baseline b128.

FIG. 89C: TCEP baseline SBS sequencing method.

FIG. 90 presents exemplary data showing a Q Score comparison betweenDMPS baseline b128 and TCEP baseline cleave reagents.

FIGS. 91A-C present exemplary data comparing two DMPS concentrations (75versus 50 mM) to a TECP standard to demonstrate improvements inlead/lag.

FIG. 92 presents exemplary data comparing two DMPS concentrations (75versus 50 mM) to a TECP standard to demonstrate improvements in averageerror rate.

FIG. 93A-C shows one embodiment of a sequencing workflow. FIG. 93A showsone embodiment of a workflow for primer hybridization. FIG. 93B shows onembodiment of a workflow for flow cell preparation. FIG. 93C shows oneembodiment of an automated workflow for sequencing by synthesis (SBS),wherein an oxidative wash (Wash 11) is used after the cleave step.

FIG. 94A shows lead/lag results when tert-butyl peroxide is used in theoxidative wash (Wash 11), as compared to lead/lag results when hydrogenperoxide is (FIG. 94B) or is not (FIG. 94C) in the oxidative wash. Leadas a function of cycle needs to be a linear function in order for thealgorithm to be able to measure an accurate number. FIG. 94C showsnon-linear lead results when the wash does not contain hydrogenperoxide. However, when the chemistry was optimized properly byincluding hydrogen peroxide (FIG. 94B) or tert-butyl peroxide (FIG. 94A)in the oxidative wash, a linear function was achieved and a reliablevalue for lead was provided by the algorithm.

FIG. 95 shows the structures for CMIT and MIT found in the commerciallyavailable reagent “Proclin300” (which is a mixture of5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT);2-Methyl-4-isothiazolin-3-one (MIT); glycol: 93-95%; modified alkylcarboxylate: 2-3%).

FIG. 96 is a graph showing a comparison of read length in DNA sequencingunder two different conditions/configurations. In one configuration(B290), a compound is used as a sequencing additive, i.e.5-Chloro-2-methyl-4-isothiazolin-3-one is utilized in the extend reagentfor the extension reaction. In the other configuration (B233), thiscompound is not utilized in the extend reagent for the extensionreaction. As can be seen from the chart, a significant improvement inread length is observed for the B290 configuration (157 cycles) ascompared to the B233 configuration (137 cycles).

FIG. 97 is a graph showing a comparison of error rate (per tile) in DNAsequencing under two different conditions/configurations. In oneconfiguration (B290), a compound is used as a sequencing additive, i.e.5-Chloro-2-methyl-4-isothiazolin-3-one is utilized in the extend reagentfor the extension reaction. In the other configuration (B233), thiscompound is not utilized in the extend reagent for the extensionreaction. As can be seen from the chart, a significant improvement inread quality (reduced degradation from the inlet to the outlet region ofthe flow cell) is observed for the B290 configuration (157 cycles) ascompared to the B233 configuration (137 cycles).

FIG. 98 is a graph showing the quality score distribution across allbases where 5-Chloro-2-methyl-4-isothiazolin-3-one is utilized in theextend reagent for the extension reaction (with Q30 reaching 70-75%).

FIG. 99 presents a schematic of one embodiment of a kit (with arrows toshow the work flow) comprising reagents as disclosed herein to performSBS sequencing, and in particular, extension reactions for SBSsequencing. Box 1 comprises containers of extension premix, wash and acleave base buffer. Box 2 comprises containers of nucleotide analogues(both labeled and unlabeled), DNA polymerase and an imaging buffer. Thiskit does not contain CMIT.

FIG. 100 presents a schematic of one embodiment of a kit (with arrows toshow the work flow) comprising reagents as disclosed herein to performSBS sequencing, and in particular, extension reactions for SBSsequencing. Box 1 comprises containers of extension premix, wash and acleave base buffer. Box 2 comprises containers of nucleotide analogues(both labeled and unlabeled), DNA polymerase, an imaging buffer and CMIT(in the form of “Proclin”) as a sequencing additive or “add-on.” Thearrows show the mixing of these components to make the extend reagent.

DESCRIPTION OF THE INVENTION

The present invention provides methods, compositions, mixtures and kitsutilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencingreactions, and in particular, sequencing reactions where deoxynucleosidetriphosphates comprising a 3′-O position capped by a disulfide-based3′-terminator group are used. In one embodiment, the present inventionprovides methods, compositions, mixtures and kits utilizing5-Chloro-2-methyl-4-isothiazolin-3-one together with deoxynucleosidetriphosphates comprising a 3′-O position capped by group comprisingmethylenedisulfide as a cleavable protecting group and a detectablelabel reversibly connected to the nucleobase of said deoxynucleoside.Such compounds provide new possibilities for future sequencingtechnologies, including but not limited to Sequencing by Synthesis (SBS)and particularly automated SBS in a flow cell. The present inventioncontemplates, as compositions of matter, the various structures shown inthe body of the specification and the figures. These compositions can beused in reactions, including but not limited to primer extensionreactions. These compositions can be in mixtures. For example, one ormore of the labeled nucleotides (e.g. such as those shown in FIG. 25)can be in a mixture (and used in a mixture) with one ore more unlabelednucleotides (e.g. such as those shown in FIG. 51). They can be in kitswith other reagents (e.g. buffers, polymerases, primers, etc.)

In one embodiment, the labeled nucleotides of the present inventionrequire several steps of synthesis and involve linking variety of dyesto different bases. It is desirable to be able to perform linker and dyeattachment in a modular fashion rather than step by step process. Themodular approach involves pre-building of the linker moiety withprotecting group on one end and activated group on the other. Suchpre-built linker can then be used to couple to apropargylaminenucleotide; one can then, deprotect the masked amine group and thencouple the activated dye. This has the advantage of fewer steps andhigher yield as compare to step-by-step synthesis.

In one embodiment, the labeled nucleotides of the present invention areused in DNA sequencing. DNA sequencing is a fundamental tool in biology.It is a widely used method in basic research, biomedical, diagnostic,and forensic applications, and in many other areas of basic and appliedresearch. New generation DNA sequencing technologies are changing theway research in biology is routinely conducted. It is poised to play acritical role in the coming years in the field of precision medicines,companion diagnostics, etc.

Sequencing by synthesis (SBS) is a revolutionary next-generationsequencing (NGS) technology, where millions of DNA molecules, single orcluster thereof can be sequenced simultaneously. The basis of thistechnology is the use of modified nucleotides known as cleavablenucleotide terminators that allow just a single base extension anddetection of the DNA molecules on solid surface allowing massiveparallelism in DNA sequencing (for comprehensive reviews: Cheng-Yao,Chen, Frontiers in Microbiology, 2014, 5, 1 [22]; Fei Chen, et al,Genomics Proteomics Bioinformatics, 2013, 11, 34-40 [5]; C. W. Fuller etal, Nature Biotechnology, 2009, 27, 1013 [2]; M. L. Metzker, NatureReviews, 2010, 11, 31 [1])—all of which are hereby incorporated byreference.

Modified nucleotides, with 3′-OH positions blocked by a cleavableprotecting group, which after incorporation into DNA primers andsubsequent detection, can be removed by chemical reaction, are the keyto the success of the SBS chemistry (Ju et al, U.S. Pat. No. 7,883,869,2011 [23]; Ju et al, U.S. Pat. No. 8,088,575, 2012 [24]; Ju et al, U.S.Pat. No. 8,796,432, 2014 [25]; Balasubramanian, U.S. Pat. No. 6,833,246,2004 [26]; Balasubramanian et al, U.S. Pat. No. 7,785,796B2, 2010 [27];Milton et al, U.S. Pat. No. 7,414,116 B2, 2008 [28]; Metzker, M. L., etal, Nucleic Acids Res, 1994, 22:4259-4267 [29]; Ju et al, Proc. Nat.Acad, Sci. USA, 103 (52), 19635, 2006 [30]; Ruparel et. al, Proc. Nat.Acad, Sci. USA, 102 (17), 5932, 2005 [31]; Bergmann et al, US2015/0140561 A1 [32]; Kwiatkowski, US 2002/0015961 A1 [33])—all of whichare hereby incorporated by reference.

There have also been attempts to develop nucleotide analogs, known asvirtual terminators, where the 3′-OH is unprotected but the bases aremodified in such a manner that the modifying group prevents furtherextension after a single base incorporation to the DNA templates,forcing chain termination event to occur (Andrew F. Gardner et al.,NucleicAcidsRes 40(15), 7404-7415 2012 [34], Litosh et al, Nuc. Acids,Res., 2011, vol 39, No. 6, e39 [35], Bowers et al, Nat. Methods, 2009,6, 593 [36])—all of which are hereby incorporated by reference.

Also disclosed were ribo-nucleotide analogs, where the 2′-OH isprotected by removable group, which prevents the adjacent 3′-OH groupfrom participating in chain extension reactions, thereby stopping aftera single base extension (Zhao et al, U.S. Pat. No. 8,399,188 B2, 2013[37]), incorporated by reference.

On the other hand, Zon proposed the use of dinucleotide terminatorscontaining one of the nucleotides with the 3′-OH blocked by removablegroup (Gerald Zon, U.S. Pat. No. 8,017,338 B2, 2011 [38]), incorporatedby reference.

Previously a cleavable disulfide linker (—SS—) has been used to attachfluorescent dye in the labeled nucleotides for use in the GeneReadersequencing. It is believed that the —SH scars left behind on the growingDNA strain after cleaving step, causes a number of side reactions whichlimit achieving a longer read-length.

It is known that —SH residues can undergo free radical reactions in thepresence of TCEP used in cleaving step, creating undesired functionalgroup, and it potentially can damage DNA molecules (Desulfurization ofCysteine-Containing Peptides Resulting from Sample Preparation forProtein Characterization by MS, Zhouxi Wang et all, Rapid Commun MassSpectrom, 2010, 24(3), 267-275 [39]).

The —SH scars can also interact with the incoming nucleotides inside theflow-cell cleaving the 3′ OH protecting group prematurely causingfurther chain elongation and thereby it can cause signal de-phasing.

The end result of the detrimental side reactions of —SH is the reductionof the read-length and increased error rates in the sequencing run.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, compositions, mixtures and kitsutilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencingreactions, and in particular, sequencing reactions where deoxynucleosidetriphosphates comprising a 3′-O position capped by a disulfide-based3′-terminator group are used. The present invention provides methods,compositions, mixtures and kits utilizing5-Chloro-2-methyl-4-isothiazolin-3-one together with deoxynucleosidetriphosphates comprising a 3′-O position capped by group comprisingmethylenedisulfide as a cleavable protecting group and a detectablelabel reversibly connected to the nucleobase of said deoxynucleoside.The present invention also provides methods, compositions, mixtures andkits utilizing thiol derivatives (alternatively mercaptol analogues) ascleave agents of the disulfide based linkers and 3′-disulfide cappinggroup of nucleotides pertinant to DNA sequencing technologies (and othertechnologies such as protein engineering) where the disulfide reductionor cleavage step is necessary. In one embodiment, the cleave agent iskitted as a solid powder add-on that the customer will add to the cleavepre-mix buffer according to sequencing workflow instructions. Moreover,the present invention provides embodiments where the thiol-containingcompounds are scavenged with an oxidative wash step. Such compoundsprovide possibilities for use in future sequencing technologies,including but not limited to Sequencing by Synthesis (SBS), includingautomated SBS in a flow cell.

The present invention, in one embodiment involves the synthesis and useof a labeled nucleoside triphosphates comprising a cleavableoxymethylenedisulfide linker between the label and nucleobase, with a3′-O group comprising methylenedisulfide as a protecting group, havingthe formula —CH₂—SS—R, in DNA sequencing (e.g. sequencing by synthesis),where the R represents alkyl group such as methyl, ethyl, isopropyl,t-butyl, n-butyl, or their analogs with substituent group containinghetero-atoms such as O, N, S etc (see FIG. 1). In one embodiment, the Rgroup may contain a functional group that could modulate the stabilityand cleavability of the 3′-O capping group, while being acceptable toDNA polymerase enzymes.

In another aspect, the invention relates to a labeled nucleosidetriphosphates comprising a cleavable oxymethylenedisulfide linkerbetween the label and nucleobase, with 3′-O positions capped by a groupcomprising methylenedisulfide wherein the nucleobases can be natural, ornon-natural bases which can form DNA duplex by hydrogen bondinteractions with natural nucleobases of the DNA templates, and that canbe 7-deaza analog of dG and dA, and 2-amino-dA. 7-deaza analogs of dAand dG can reduce the formation of DNA tertiary structures due to thelack of 7-N atom. It is envisioned that in one embodiments, suchnucleosides could potentially improve DNA sequencing read-length byenhancing DNA templates and polymerase interaction. It may also bepossible that the 2-amino-dA can increase DNA duplex stability due toits ability to form more stable 3 hydrogen bonds with its compimentarybase (rather than 2 bond in natural state), therefore, it can reduce therisk of losing DNA primers during sequencing run (A Jung et all, Mol.Pathol., 2002, 55 (1), 55-57 [40]; 2-amino-dATP: Igor V. Kutyavin,Biochemistry, 2008, 47(51), 13666-73 [41]).

In another embodiment, said nucleotides may have detectable reportermolecules, such as fluorescent dyes linked to nucleobases via cleavablelinker —OCH₂SS—. Labeled nucleotides, where the —OCH₂—SS— group isdirectly attached to the nucleobases and the use thereof as cleavablelinker are not known in prior-art. Contrary to the traditional, widelyused disulfide linkers (—SS—), this class of cleavable linker(—OCH₂—SS—) leaves no sulfur trace on the DNA molecule, cleanlyconverting it to —OH group by rapid hydrolysis of the resultingintermediate, —OCH₂—SH, after reductive cleavage. Because of this, suchlinkers may be better alternatives to the traditional disulfide linkers.In tranditional disulfide based linkers (—SS—), the resulting thiolgroup (—SH) can undergo side reactions when cleaved by reducing reagentssuch as TCEP as presented in the following FIG. 4 (Ref: Desulfurizationof Cysteine-Containing Peptides Resulting from Sample Preparation forProtein Characterization by MS, Zhouxi Wang et all, Rapid Commun MassSpectrom, 2010, 24(3), 267-275 [39]).

In another embodiment, the reporter groups may be attached to thepyrimidine bases (dT, dC) at 5-C position and to purine bases (dA, dG)at 7-N of natural bases, or 7-C of de-aza analogs.

In another embodiment, the structure of the labeled nucleotides may beas shown in FIG. 5, where the spacer of the cleavable linker includesthe propargyl ether linker. The nucleobases with progargyl ether can besynthesized following prior arts of chemical synthesis. The L₁ and L₂represent chemical spacers, and substituents R₁, R₂, R₃ and R₄ are groupof atoms that modulate stability and cleavability to the cleavablelinker. They can be hydrogen atom, geminal dimethyl, or any alkyl,phenyl, or substituted alkyl group, such as methyl, ethyl, n-butyl,phenyl, etc. They may also contain a hydrocarbon chain with —O, ═O,NH,—N═N, acid, amide, poly ethyleneglycol chain (PEG) etc. The label on thenucleotides may be fluorescent dyes, energy transfer dyes, radioactivelabel, chemi-luminiscence probe, heptane and other form of label thatallows detection by chemical or physical methods.

In another embodiment, the structure of the labeled nucleotides may beas shown FIG. 6. The spacers of the cleavable linker include thepropargylamine linker. Again, the L₁ and L₂ represent spacers, andsubstituents R₁, R2, R3 and R₄ are group of atoms that provide stabilityand modulate cleavability of the linker as described earlier. They maybe hydrogen atoms, alkylgroups such as methyl, ethyl and othersubstituted groups or their salts. Geminal dialkyl group on the α-carbonof the cleavable disulfide linker (e.g. germinal dimethyl analogueaccording to the following structure:

provides better stability to the linker allowing modular synthesis oflabeled nucleotides. It presumably prevents disproportional reactionsprevalent among disulfide based organic compounds. It also adds greaterhydrophobicity to the linker which helps the synthesis and purificationof labeled nucleotide analogues [42-44]. The gem dimethyl functionalitypresent in the linker is believed to not only serve to stabilize thedisulfide bond electronically, but also prevents disulfide exchange fromoccurring both inter- and intra-molecularly, likely via sterric effects.It has been demonstrated that in the presence of cystamine, thedisulfide functionality on the terminator participates in disulfideexchange, while linkers equipped with gem dimethyl groups do not. Thelinker study in FIG. 42 compares linkers with and without the gemdimethyl group. As can be seen from this study, linkers G and L withoutthe gem dimethyl group quickly exchange with cystamine leading todegradation of the product. As expected, this phenomenon is not observedwith our chosen linker A, nor with analogous linker B. In addition,since the labelled nucleotides contain two disulfides, one on theterminator and one on the linker portion of the molecule, it is believedthat this stabilizing effect prevents scrambling between the dye and theterminator from occurring. This stability is important to performance ofour nucleotides in sequencing.

In another embodiment, the structure of the labeled nucleotides may beas in FIG. 7. The spacer of the cleavable linker include the methylene(—(CH₂)_(n)— directly attached to the nucleobases at 5-position forpyrimidine, and at 7-de-aza-carbon for purines. This linker may bemethylene (n=1) or polymethylene (n>1) where after cleavage, the linkergenerates —(CH₂)OH group at the point of attachment on the nucleobases,and where the L₁ and L₂ represent spacers, and substituents R₁, R₂, R₃and R₄ are group of atoms that provide stability to the cleavable linkeras described earlier.

In another embodiment, the invention relates to synthetic methods forthe nucleotides claimed. The capping group and linker may be synthesizedmodifying prior arts described For example, the unlabeled dT analog(compound 5) can be synthesized as shown in FIG. 8.

In one embodiment the invention involves: (a) nucleoside triphosphateswith 3′-O capped by a group comprising methylenedisulfide (e.g. of theformula —CH₂—SS—R) as a cleavable protecting group (see FIG. 1); and (b)their labeled analogs (see FIG. 2), where labels are attached to thenucleobases via a cleavable oxymethylenedisulfide linker (—OCH₂—SS—).Such nucleotides can be used in nucleic acid sequencing by synthesis(SBS) technologies. General methods for the synthesis of the nucleotidesclaimed are also described.

In one embodiment, as shown in FIG. 1, the general structures ofunlabeled nucleotides have the 3′-O group protected by a groupcomprising methylenedisulfide with a common structure —CH₂—SS—R, wherethe R can be regular alkyl or substituted alkyl groups such as -Me, -Et,-nBu, -tBu, —CH₂CH₂NH₂, —CH₂CH₂NMe etc., and B, can be natural ornon-natural nucleobases. Some specific examples of non-naturalnucleobases are 7-deaza dG and dA, 2-amino-dA etc.

In FIG. 2, the general structures of labeled analogs are shown with 3′-Oprotected by a group comprising methylenedisulfide as in FIG. 1, inaddition to that a detectable reporter (label) such as fluorophore isattached to the nucleobases via a cleavable linker having a generalstructure -L₁₋OCH₂—SS-L₂-. L₁ represents molecular spacer that separatesnucleobase from the cleavable linker, while L₂ between cleavable linkerand the label, respectively. Both L₁ and L₂ can have appropriatefunctional groups for connecting to the respective chemical entitiessuch as —CO—, —CONH—, —NHCONH—, —O—, —S—, —C═N, —N═N—, etc. The labelmay be fluorophore dyes, energy transfer dyes, mass-tags, biotin,haptenes, etc. The label may be different on different nucleotides fordetection of multiple bases simultaneously, or the same for step-wisedetection of spatially separated oligonucleotides or their amplifiedclones on solid surface.

In one embodiment, the invention relates to a new class of nucleotidethat has 3′-O capped with —CH₂—SS—R group and a label attached to thenucleobase through a cleavable linker having a general structure—O—CH₂—SS—. Such capping group and linker can be cleanly cleavedsimultaneously by single treatment with TCEP or related chemicalsleaving no sulfur traces on the DNA molecules.

This class of nucleotides may be stable enough to endure the relativelyhigh temperature (˜65° C.) necessary for nucleotide incorporation ontothe DNA templates catalyzed by thermo active polymerases, yet labileenough to be cleaved under DNA compatible conditions such as reductionwith TCEP etc. In some embodiments, cleavage may be accomplished byexposure to dithiothreitol.

The nucleotide when exposed to reducing agents such as TCEP de-cap the3′-O protection group via step-wise mechanism shown in FIG. 3, thusrestoring the natural state of the 3′-OH group. TCEP and its analogs areknown to be benign to bio-molecules which is a pre-requisite forapplication in SBS.

In one embodiment, the invention relates to a generic universal buildingblocks structures comprising new cleavable linkers, shown in FIG. 28.PG=Protective Group, L1, L2-linkers (aliphatic, aromatic, mixed polaritystraight chain or branched). RG=Reactive Group. In one embodiment ofpresent invention such building blocks carry an Fmoc protective group onone end of the linker and reactive NHS carbonate or carbamate on theother end. This preferred combination is particularly useful in modifiednucleotides synthesis comprising new cleavable linkers. A protectivegroup should be removable under conditions compatible with nucleicacid/nucleotides chemistry and the reactive group should be selective.After reaction of the active NHS group on the linker with amineterminating nucleotide, an Fmoc group can be easily removed using basesuch as piperidine or ammonia, therefore exposing amine group at theterminal end of the linker for the attachment of cleavable marker. Alibrary of compounds comprising variety of markers can be constructedthis way very quickly.

In one embodiment, the invention relates to a generic structure ofnucleotides carrying cleavable marker attached via novel linker, shownin FIG. 29. S=sugar (i.e., ribose, deoxyribose), B=nucleobase, R═H orreversibly terminating group (protective group). Preferred reversiblyterminating groups include but are not limited to: Azidomethyl (—CH₂N₃),Dithio-alkyl (—CH₂—SS—R), aminoxy (—ONH₂).

In one embodiment, the present invention contemplates a thiol reagentcomprising at least two or more thiol groups either at a vicinalposition (e.g., n1=1) or separated by at least one hydrocarbon bond(e.g., n1=>1), which further attached to anionic functional group (R)with hydrocarbon spacer (e.g., n2=>1). See, FIG. 79.

In one embodiment, the present invention contemplates compounds with athiol group (—SH) that is adjacent to a functional group (R1) including,but not limited to, a halogen, an amine, a phosphate, a phosphonate, aphosphoric acid, a carboxylic acid, a sulfoxide and/or a hydoxyl etc.,which could modulate the pKa of the functional group. See, FIG. 80.

DNA sequencing plays a role as a useful analytical tools in modembiotechnology. For example, new generation DNA sequencing (NGS) isrevolutionizing in the studies of genetics, diagnostics and basicbiomedical research. Detailed reviews on current NGS technologies areprovided in M. L. Metzker, Nature Reviews 2010, 11, 31, and C. W. Fulleret al., Nature Biotechnology 2009, 27, 1013.

One well-known sequencing method is the sequencing-by-synthesis (SBS)where many DNA templates comprising nucleoside triphosphates aresequenced simultaneously during enzymatic synthesis of complementarystrains of DNA molecules. According to this method, the nucleosidetriphosphates are reversibly blocked by a 3′-OH-protecting groupincluding, but not limited to, acetyl, phosphates, carbonates,azidomethyl, nitro etc. In one embodiment, the nucleoside triphosphatecomprises a label at the base via a cleavable linker.

Although it is not necessary to understand the mechanism of aninvention, it is believed that sequencing-by-synthesis (SBS) largelydepends on an ability to achieve a longer read length and a betteraccuracy. It is also believed that the use of a 3′-OH capping group anda cleavable linker can be removed under DNA compatible conditions. Forexample, the present invention contemplates reversible nucleotideterminators where a 3′-OH group is capped with alkyl methylenedisulfide(e.g., R—SS—CH₂—) and a cleavable linker having the same functionalmoiety (e.g., —OCH₂—SS—). For simultaneous cleavage of both, a DNAcompatible cleave reagent is preferred. Unfortunately, many reducingagents are detrimental to DNA molecules and often are too harsh to thesolid surface of the flow cell, and difficult to wash away from theflow-cell.

There are a number of reducing agents are known to cleave disulfidebonds. Examples include, but are not limited to, TCEP, DTT, glutathione,borohydride etc. These reducing agents are widely used in proteinengineering and anti-body chemistry. But such compounds can also havecertain limitations not only due to slower reaction rate, but alsodifficulty in complete removal in a wash step from the flow cell whenapplied to NGS. Further, the reducing power of thiol-based compoundsdepends on the pH of the medium. It is known that at higher pH, thethiol group (RSH) undergoes ionization to form thiolate (RS—), which canspeed up the reaction rate, by many times. But at higher pH, DNAmolecules and the surface can undergo hydrolytic damage, therebylimiting its application. Therefore, in one embodiment, thiol compoundsthat can reduce disulfide at reduced pH, so that it is compatible toboth DNA and the flowcell surface, are preferred for application in SBSchemistry.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the thiol compounds shown in FIGS. 79 and80 can undergo ionization to form thiolate at a relatively low pH due tothe presence of adjacent functional group (i.e., for example, adjacent—SH and electron-withdrawing R1) via neighboring group participation.Consequently, they could reduce the disulfide bonds at relatively lowerpH and can have increased reaction rate than those of standard thiols.Furthermore, the neighboring group participation of thiol at anα-position via anchimeric assistance can speed up the second step of thereduction process due to formation of intra-molecular disulfide bond.See, FIG. 81. as shown in FIG. 3. This reaction is not believed toundergo a nucleophilic attack by another thiol molecule to form sulfidemolecule as currently believed in the art. “Thioldisulfide interchange,The Chemistry of Sulphur Containing Functional Group” Chapter 13, p633-658, by Rajeeva Singh and George M. Whitesides). Due to negativelycharged pending group (R=sulfonate, phosphonate, carboxylate etc) it isbelieved that molecules are expected to be non-sticky to the surface,thereby making them easily washable from the flow-cell. The data hereinpresents a cleavage study of a 3′ disulfide capped with an attacheddisulfide linker with thiol-based compounds that showed a completecleavage of both the capping group and the linker in less than ten (10)minutes at 65° C. in TRIS buffer (pH 8.5). Interestingly, the completecleavage was not observed in the presence of dithiosuccinate. See, FIG.82 and Table 1.

TABLE 1 Dithiol Compound Cleavage Of Nucleosides Reaction Time &Compound Compound Compound Cleaving Agent Conditions Temperature A [%] B[%] C [%] Dithiosuccinic acid Compound A- 10 min @ 65 0 50 50Dimercaptopropanesulfonate 0.2 mM, degree C. 0 0 100 L-Cysteine Cleaveagent- 0 0 100 DTT 20 mM, buffer- 0 0 100 Cysteamine 200 mM TE, pH 0 0100 8.5

The structures of these cleave reagents are shown. See, FIG. 83. Thecleavage products were then characterized by liquid chromatography/massspectrometry ((LC/MS). See, FIG. 84. Compounds A, B and C are shown inFIG. 82.

In one embodiment, the present invention contemplates a methodcomprising a cleaving step comprising a cleave agent capable of cleavinga methyl-disulfide 3′-OH protected nucleotide in a high pH nucleophiliccondition. In one embodiment, the cleave agent is a di-mercaptancompound. In one embodiment, the cleaving step occurs in a sequencing bysynthesis (SBS) step. In one embodiment, the cleaving step is followedby a wash step comprising oxidative scavenging to effectively removeresidual cleave agent.

Although it is not necessary to understand the mechanism of aninvention, it is believed that such oxidative scavenging unlocks theefficiency of di-mercaptan based cleaving and enables sequencing withgood performance, in particular with respect to reduction of lead andsignificantly improved raw error rate and longer read length. It isfurther believed that such effects may be due to, for example: (1) moreefficient cleave reaction under high pH conditions (e.g. 9.0 to 10.0,and preferably 9.5) due to formation of more reactive nucleophiliccleaving species; and/or (2) effective scavenging of cleave reagent thatis retained in the flow cell likely via surface adsorption mechanisms(e. g., chelation to iron-containing surfaces such as silicate, titania,magnetic bead ferrite). For example, such cleave residual can build upin a flow cell leading to carry over into the subsequent extension stepthus causing premature de-protection of the 3′-OH moiety and impairingsingle base incorporation with very significant detriment to sequencingperformance. In one embodiment, a di-mercaptan species as disclosedherein may be di-mercaptan propane sulfonate (DMPS), also referred to asdimercaptopropanesulfonate. In other embodiments, a di-mercaptan cleaveagent may include, but is not limited to, sodium2-mercaptoethanesulfonate (MESNA) and/or glutathione. In one embodiment,DMPS has the following structure:

In one embodiment, the present invention contemplates a methodcomprising the steps of cleaving and washing thar are compatible withSBS sequencing with compounds including, but not limited to, DMPS, MESNAand/or glutathione. In one embodiment, the cleaving step comprises DMPSin CHES buffer at pH 9.5. In one embodiment, the washing step comprisesH₂O₂ in TRIS buffer at pH 8.5. In one embodiment, the washing step mayfurther comprise an oxidative scaveenging agent including, but notlimited to, H2O2, tert-butyl peroxide and/or NaBO3.

In one embodiment, 30% and 3% are concentration of H2O2 stocks goinginto the add-on. In a preferred embodiment, the final concentration ofH2O2 in the wash mix is H2O2 0.3%.

In one embodiment, the present invention contemplates a kit comprisingat least one container and a set of instructions for performing SBSsequencing. In one embodiment, the kit further comprises at least onefirst container comprising an extension premix buffer. In oneembodiment, the kit further comprises at least one second containercomprising a cleave base buffer. In one embodiment, the kit furthercomprises at least one third container comprising a wash buffer premix.In one embodiment, the kit further comprises at least one containercomprising a wash buffer. In one embodiment, the kit further comprisesat least one container comprising an extension add-on reagent. In oneembodiment, the extension add-on reagent is A2 (PMA). In one embodiment,the extension add-on reagent is B2 (PMB). In one embodiment, the kitfurther comprises at least one container comprising a DNA polymeraseenzyme. In one embodiment, the kit further comprises at least onecontainer comprising an imaging buffer. In one embodiment, the kitfurther comprises at least one container comprising a wash add-onreagent. In one embodiment, the wash add-on reagent is hydrogen peroxide(H2O2). In one embodiment, the hydrogen peroxide is in a stock solutionat a concentration of between one percent and five percent, and morepreferably three percent (3%) that is later diluted to a lowerconcentration for the wash. In one embodiment, the kit further comprisesat least one container comprising a cleave-solid add-on reagent. In oneembodiment, the cleave-solid add-on reagent is di-mercaptan propanesulfonate (DMPS).

In one embodiment, the kit comprises a cleave reagent formulationcomprising DMPS (75 mM), CHES (300 mM), NaCl (300 mM) at pH 9.5.

In one embodiment, the kit comprises a Cleave 1 Add-on and/or a Cleave 2Add-on.

In one embodiment, the kit comprises a frozen liquid (e.g., (−20° C.)cleave reagent add-on comprising concentrated DMPS in H2O or H2O/NaCl.

In one embodiment, the kit comprises a solid cleave reagent add-oncomprising DMPS+NaCl

In one embodiment, the kit comprises a liquid (+2-8° C.) cleave reagentadd-on comprising DMPS as a neat reagent.

In one embodiment, the kit comprises a wash reagent formulationcomprising TRIS at pH 8.8, H2O2 (0.3%) and EDTA (1%).

In one embodiment, the kit comprises a liquid (2-8° C.) wash reagentadd-ons comprising H₂O₂ (3.0%). In one embodiment, the kit providesinstructions to dilute this to a lower concentration (e.g. 0.3%).

In one embodiment, the kit comprising improved stability liquid (2-8°C.) wash reagent add-ons including, but not limited to, TBHP, APS and/orNaBO3.

In one embodiment, the present invention contemplates cleave reagentsthat have been developed to function under SBS sequencing conditions. Inone embodiment, such a cleave reagent is di-mercaptan propane sulfonate.

As described herein, the cleave reagents are designed to meet thefollowing SBS criteria: i) average read length of approximately 85 bp;ii) average raw error rate of approximately five percent (5%); iii) beadretention that is greater than ninety percent (>90%); and/or iv) a dwelltime of approximately one and one-half times (1.5×). These criteria wereevaluated for performance improvement and optimization indicatorsincluding, but not limited to: i) buffer formulation; ii) DMPStitration; and/or cleave and iron scavenging condition optimization. Thedata discussed below demonstrate that at least one cleave reagent,di-mercapto propane sulfonate (DMPS), meets and/or exceeds thesecriteria. Although it is not necessary to understand the mechanism of aninvention, it is believed that SBS sequencing with a DMPS-based cleavereagent is enabled by nucleophilic conditions at a cleave stepcomprising CHES buffer at pH 9.5 followed by a post-cleave washing stepcomprising an oxidative scavenging condition, using for example,hydrogen peroxide (H2O2)

Candidate cleave reagents including but not limited to, di-mercaptoreagents, mono-mercapto reagents and/or helico reagents were screened byhigh performance liquid chromatography. The screening configuration wasperformed on a GeneReader and aside from a candidate cleave reagentincluded reagents such as oxidative scavengers, capping agents and/orchelators. This screening assay observed optimization of the extendingand washing steps using an SBS sequencing method including reagents suchas, DMPS/MESNA, inorganic compounds and/or enzymatic compounds.Additionally, off-GeneReader assays including, but not limited toclearance, activity and/or FC integrity were also performed. Theobservations to optimize the cleaving step included, but are not limitedto, pH, concentration, buffer type/counterion, dwell-time and/ortemperature.

A DMPS baseline was constructed using commercially available DNAstandards (Acrometrix) using an AIT for 107 cycles. Preliminaryobservations found that the inclusion of an oxidative scavenger in thewashing step controlled lead significantly and that the pH level playeda role in mediating the overall cleave efficiency. Subsequentverification studies identified that a preferred DMPS baselineconfiguration comprises a DMPS cleave reagent in CHES 300 mM pH 9.5followed by a dilute H2O2, wherein the GeneReader is operated at 65° C.for cleave; 1× dwell (TAT requirement compliant) and a 3× fluidic wash.See, Table 2.

TABLE 2 DMPS Baseline SBS Sequencing Configuration (Acrometrixstandards) Cockpit % Bead % % @ AVG Raw Filtered Cleave Configuration(GDPn) Code Ret Perfect Q25*** RL Error Error Lead Lag DMPS CHES 300 mMpH 9.5 (GDP4) b128   97%   52%   82% 84  4.37% 1.00% 0.58** 0.07 SDev0.80% 5.60% 2.60% 2.28  0.80% 0.20% 0.02 0.01 DMPS CHES 50 mM pH 9.5(GDP3) b124   95%   44%   82% 80  7.10% 1.30% n.m.* 0.12 SDev 2.67%2.89% 1.34% 3.02  0.07% 0.05% 0.02 DMPS CHES 50 mM pH 9 (GDP3) b121  95%   45%   80% 74  9.10% 1.92% n.m.* 0.12 SDev 1.78% 2.45% 1.78% 1.98 0.03% 0.03% 0.02 DMPS TRIS pH 8.5 (GDP3) b77   97%   40%   75% 6517.03%  3.8% n.m.* 0.12 SDev 1.90% 2.01% 1.89% 1.89  0.07% 0.03% 0.02New SBS TCEP Baseline (GDP5) b34   97%   67%   85% 89  3.25% 0.60% 0.490.12 SDev 1.50% 2.31% 1.56% 1.56  0.03% 0.03% 0.03 0.02

These data showed that DMPS CHES 300 mM at pH 9.5 provided a measurablelead and that the raw error rate and average read length met the abovedescribed criteria.

The data presented herein demonstrates that the raw error rate improvesas a function of configuration evolution. See, FIG. 88. For example,having DMPS in the cleave step without the H₂O₂ in the wash step doesnot allow sequencing past 25 cycles (data not shown). Consequently, thedata suggests that SBS sequencing with both DMPS and H₂O₂ enables betterperformance because of the higher pH as seen when comparing; i) poorsequencing performance (pH 8.5; red curve); ii) high quality performance(pH 9.5; blue curve); and iii) TCEP benchmark (green curve). FIG. 88.

The data presented herein demonstrate that the lead/lag comparison isimproved (when compared to a 8.5 pH DMPS TRIS cleave reagent) when usingeither a DMPS baseline b128 or a TCEP baseline SBS sequencing method.See, FIG. 89A cf FIG. 89B and FIG. 89C. It can be seen that lead isnear-linear in the high pH configuration and comparable to TCEP EarlyAccess Baseline. Further, a Q Score analysis show that AVG values forDMPS baseline b128 are about 4 Q scores lower than TCEP Early AccessBaseline. See, FIG. 90.

A secondary analysis benchmarking analysis found that the average FP for0.5% AF calls is 2 as compared to null for TCEP Early Access. See, Table3.

TABLE 3 Secondary Analysis Benchmarking Configuration Q85 Coverage >200×TP FN FP @ 0.5% calls b34 (TCEP Early Access)001146_MAN08_RA01.BC3.fastq 26 100% 68 5 (0) 0001146_MAN08_RA01.BC4.fastq 26 100% 68 5 (0) 0001154_MAN08_RA01.BC3.fastq 26 100% 68 5 (0) 0001154_MAN08_RA01.BC4.fastq 26 100% 68 5 (0) 0 AVG FP = 0 b124000215_man09_RA01.BC3 23 100% 67 6 (1) 4 000215_man09_RA01.BC4 23 100%67 6 (1) 5 000879_Man08_clones_RA01.BC3 26  89% 65 8 (3) 35000879_Man08_clones_RA01.BC4 22 100% 67 6 (1) 8 000965_man09_RA01.BC3 25100% 66 7 (2) 38 000965_man09_RA01.BC4 22 100% 67 6 (1) 8 AVG FP = 16b128 (DMPS Baseline) 000243_Man11_RA01.BC3.fastq 24 100% 67 6 (1) 0000243_Man11_RA01.BC4.fastq 24 100% 67 6 (1) 0000988_MAN09_RA01.BC3.fastq 22 100% 66 7 (2) 1000988_MAN09_RA01.BC4.fastq 22 100% 66 7 (2) 4001038_Man11_RA01.BC3.fastq 24 100% 67 6 (1) 0001038_Man11_RA01.BC4.fastq 24 100% 67 6 (1) 3001039_MAN09_RA01.BC3.fastq 24 100% 67 6 (1) 1001039_MAN09_RA01.BC4.fastq 24 100% 67 6 (1) 4 AVG FP = 2

DMPS baseline reproducibility was tested during eight (8) SBS runs usingDMPS in CHES 300 mM pH 9.5 followed by a dilute H2O2-containing washoperated at 65° C. cleave, a 1× dwell that was TAT requirementcompliant, and a 3× fluidic wash. See, Table 4.

TABLE 4 DMPS Baseline Reproducibility % Bead % % @ AVG Raw Fittered RunDate/b128 GR Ret Mapped Q25 RL Error Error Output Lead Lag Dec. 16, 20168.1 97% 28% 79% 85 4.17% 1.10% 1.31 0.53 0.04 Dec. 16, 2016  8.17 95%27% 85% 86 3.83% 0.85% 1.26 0.58 0.05 Dec. 16, 2016  8.31 97% 30% 84% 843.88% 0.85% 1.41 0.61 0.02 Dec. 16, 2016  8.26 97% 29% 84% 84 4.07%0.85% 1.37 0.57 0.03 Dec. 19, 2016 8.1 97% 26% 81% 85 4.56% 1.10% 1.290.54 0.05 Dec. 19, 2016  8.17 97% 29% 84% 86 3.95% 0.89% 1.38 0.58 0.04Dec. 19, 2016  8.31 95% 29% 84% 85 4.39% 1.02% 1.38 0.55 0.05 Dec. 19,2016  8.26 97% 30% 84% 86 4.12% 0.95% 1.49 0.57 0.04 AVG 97% 29% 83% 854.12% 0.95% 1.36 0.56 0.04 SDev 0.93%   1.34%   1.93%   0.83 0.25% 0.11%0.07 0.026  0.011

The data shows comparable performance with an approximate % standarddeviation across all KPI's.

Further data a demonstrated the ability of either increasing the cleavereagent pH to pH 10 and/or decreasing DMPS concentrations to furtherimprove SBS sequencing methods. It was observed that a cleave reagent@pH 10: does NOT impact bead retention (97%); while there appears nosignificant impact on performance (data not shown). A 50 mMconcentration of DMPS (b132) was compared to a 75 mM concentration ofDMPS (b128). See, Table 5.

TABLE 5 Comparision of DMPS b132 (50 mM) To DMPS b128 (75 mM)Configuration/ % Bead % % AVG Raw Filtered Run Date GR Ret Mapped @ Q25RL Error Error Output Lead Lag b128-Dec. 16, 2016 8.17 95% 27% 85% 863.83% 0.85% 1.26 0.58 0.05 b12-Dec. 9, 2016 8.17 97% 29% 84% 86 3.95%0.89% 1.38 0.58 0.04 b132-Dec. 21, 2015 8.17 97% 84% 84% 88 3.75% 0.80%1.51 0.51 0.09

The data show that decreased DMPS concentration provides a noticeableimprovement of lead and suggest improvements in average error rate andread length. See. FIGS. 91A-C, and FIG. 92. It is also suggested thatlower DMPS concentration will also support a more advantageous COGS.

In summary, the above data shows that a high pH cleave reagent, such asDMPS, provides an SBS sequencing method comprising: i) an average readlength of approximately 85 bp or more; ii) and average raw error rate ofapproximately 4% or less; iii) bead retention of approximately 97% ormore; and iv) a 1× dwell time. Furthermore, identified residualgap-to-product requirements demonstrated an average raw error ragteranging between approximate 4% to 2.5% and an FP @ 0.5% calls rangingbetween 2 to 0.

A comparison of the DMPS b132 configuration with the TCEP configurationswas prepared. See, Table 6.

TABLE 6 Performance comparison for different configurations based onAcrometrix on AIT (107 cycles) GR1.0 Legacy n (runs) = 35 % % @ AVG RawFiltered Output Configuration GDP5 Mapped Q25 RL Error Error (Gb) LeadLag Average 35.9% 85.5% 80 2.75% 0.49% 1.08 0.36 0.12 SDev  3.3%  0.7%1.4 0.35% 0.03% 0.10 0.03 0.02 GR1.1 Legacy n (runs) = 4 ConfigurationGDP4 Average  32% 86% 95 2.11% 0.49% 1.10 0.26 0.13 SDev 1.3% 0.5% 1.10.27% 0.05% 0.10 0.05 0.03 New SBS AMP 2016 n (runs) = 11 TCEPConfiguration GDP4 Average 31% 86% 89 3.25% 0.55% 1.05 0.59 0.08 SDev 3%  1% 5.3 0.27% 0.03% 0.06 0.06 0.06 New SBS EA 2016 n (runs) = 9 TCEPConfiguration GDP5 Average   37%   88% 95 2.33% 0.60% 1.25 0.49 0.12SDev 1.50% 1.14% 0.6 0.15% 0.04% 0.03 0.05 0.01 New S8S DMPS 2016 n(runs) = 5 Configuration b132 GDP4 Average 35.9%   84% 88 3.60% 0.81%1.01 0.51 0.09 SDev 0.40% 0.80% 0.55 0.12% 0.05% 0.03 0.05 0.01

The data show that DMPS b132 configuration (as a TECP replacement) showsKPI's that are very close to TECP results using the second generation(“Early Access”) configuration.

In one embodiment, the present invention contemplates a methodcomprising a nucleic acid sequence wherein the nucleic acid templateused for sequencing is derived from a tissue biopsy. In one embodiment,the tissue biopsy comprises a liquid tissue biopsy. Although it is notnecessary to understand the mechanism of an invention it is believedthat a liquid tissue biopsy is compatible with SBS sequencing methods inorder to sequence a nucleic acid sequence within the liquid tissuebiopsy. See, Table 7.

TABLE 7 Platform KPIs For DMPS b132 SBS Configuration Liquid BiopsySample Type/Configuration (New SBS Chemistry)-Early Access Sample Rel.Sequencing Barcode Abundance Cycles GenePanel/Library BC1 1/6

107 WT Horizon cfDNA BC2 1/6

107 WT Horizon cfDNA BC3 1/6

107 AIT (HD780 % 1 cfDNA) BC4 1/6

107 AIT (HD780 % 1 cfDNA) BC5 1/6

107 AIT (HD780 % 5 cfDNA) BCS 1/6

107 AIT (HD780 % 5 cfDNA) New SBS DMPS LB Configuration b132 Average  33%   85% 90 3.33% 0.78% 1.58 0.45 0.11 SDev 0.40% 0.80% 0.55 0.12%0.05% 0.03 0.05 0.01 New SBS EA 2016 TCEP Configuration Average   38%  88% 95 2.33% 0.60% 1.71 0.49 0.12 SDev 1.50% 1.14% 0.6 0.15% 0.04%0.03 0.05 0.01

indicates data missing or illegible when filed

It can be seen that the platform KPI's for DMPS b132 are very close toTECP Early Access KPI's. The results of the liquid biopsy sequencingruns are shown. See, Table 8.

TABLE 8 Liquid Biopsy Panel Specifications Parameter Detail Panel Size12 Genes/16.7 kb Insight Size 732 unique variant positions Amplicons 330Average Amplicon Size 134 bp DNA Input 10 ng × 4 Throughput 10-40samples per run Variant Frequency Cutoff 1% Variant InsightCoverage >500×: 99.973% (KPI 98%) (6-plex) >200×: 99.984% (KPI 99%)Variant Insight Coverage >500×: 99.843% (10-plex in-silico) >200×:99.978%

The data shows outstanding coverage at both amplicon and variant insightlevels.

The accuracy of sequencing nucleic acids in liquid biopsy samples isshown below in a series of tables demonstrating the sensitivity andselectivity when using a commercially available tumor panel (e.g.,Actionable Insights Tumor Panel, Qiagen). See, Tables 9-11.

TABLE 9 Summary: Performance at 0.5% Frequency Cut-Off and AQ >20 UsingBioX KPI's 200× 500× coverage coverage Sensitivity Specificity PrecisionLAF Actionable Insights Tumor Panel 99% 98% 99% 99% 95% 10% (ATP) V2.0(FFPE) Actionable Insights Tumor Panel 99% 98% 95% 95% 90%  5% (ATP)V2.0 (FFPE) Actionable Insights Tumor Panel 99% 98% 90% 90% 90%  1%(ATP) V2.0 (plasma)

TABLE 10 Detailed: Performance at 0.5% Frequency Cut-Off and AQ >20Total Average % >500× >1000× average number read mapped read read errorrate All All All Sample name of reads length reads coverage coverage %Sensitivy Specificity Precision Legacy GR1.0 Average 2,379,003 80.5186.50 99.55 98.06 0.40 0.94 1.00 0.84 (n = 2) Early Access Average2,364,234 94.24 94.62 100.00 99.94 0.43 1.000 0.9999 0.9877 WAL Runs (n= 18) Early Access Average 2,008,043 93 94.53 99.94 99.77 0.44 0.96880.9993 0.9410 MAN Runs (n = 4) b132 44206_000135_3_BC03 2,724,496 91.2493.79 100 99.87 0.65 1 1 0.89 (DMPS 44206_000135_4_BC04 2,600,349 91.2393.73 100 99.74 0.65 1 1 0.89 Configuration) 44241_000131_3_BC032,576,597 91.98 93.04 100 99.87 0.65 1 1 1 44241_000131_4_BC04 2,569,21991.58 88.13 99.87 99.74 0.65 1 1 1 44202_000134_3_BC03 2,706,162 89.4694.49 100 99.87 0.65 0.88 1 1 44202_000134_4_BC04 2,576,711 89.46 94.49100 99.74 0.65 1 1 1 50138_000130_3_BC03 2,670,057 88.74 94.98 100 99.870.65 0.88 1 1 50138_000130_4_BC04 2,554,371 88.78 94.99 100 99.74 0.65 11 1 Average 2,622,245 90 93.47 99.98 99.81 0.62 0.9700 1.0000 0.9725

KPIs refers to secondary analysis specifications for coverage (200× and500×) and variant call (precision, sensitivity, specificity). All b132runs (n=8) have better sensitivity, specificity and precision comparedto generation 1 (“Legacy”) Nucleotides GR 1.0 Baseline and generation 2(“Early Access”) Configurations. All KPI's (sensitivity, specificity,precision) and coverage requirement for 10-plex are met.

We also plexed a number of libraries (eight) together in the liquidbiopsy (LB) sample pools.

TABLE 11 Degree of Plexing on 1% LB sample (n = 8) Total TotalAverage >200× >500× >1000× average number number read read read readerror rate All All All All All All Plexing of reads of bases lengthcoverage coverage coverage % TPs FPs FNs Sensitivy Specificity Precision 6-plex 2,622,245 234,184,033 90.309 100.000 99.984 99.805 0.616 7.7500.250 0.250 0.970 1.000 0.973  8-plex 1,966,684 175,634,383 90.308100.000 99.935 99.659 0.616 7.875 0.375 0.125 0.985 1.000 0.958 10-plex1,573,347 140,504,711 90.305 100.000 99.838 98.853 0.621 7.625 0.1250.375 0.955 1.000 0.985 12-plex 1,311,123 177,093,757 90.313 100.00099.805 98.320 0.623 7.750 0.250 0.250 0.970 1.000 0.973 16-plex  970,231 86,653,104 90.315  99.935 99.675 97.719 0.625 7.750 0.375 0.250 0.9701.000 0.959 20-plex  786,674  70,258,227 90.314  99.935 98.805 95.9580.629 7.500 0.125 0.500 0.940 1.000 0.985 24-plex  655,561  58,542,08790.305  99.838 98.320 92.675 0.631 7.250 0.500 0.750 0.908 1.000 0.936 *Values are average over 8 HD780 1% cfDNA barcodes

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Synthesis of3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine(2)

5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine (1) (2.0 g, 5.6 mmol)was dissolved in a mixture consisting of DMSO (10.5 mL), acetic acid(4.8 mL), and acetic anhydride (15.4 mL) in a 250 ml round bottom flask,and stirred for 48 hours at room temperature. The mixture was thenquenched by adding saturated K₂CO₃ solution until evolution of gaseousCO₂ was stopped. The mixture was then extracted with EtOAc (3×100 mL)using a separating funnel. The combined organic extract was then washedwith a saturated solution of NaHCO₃ (2×150 mL) in a partitioning funnel,and the organic layer was dried over Na₂SO₄. The organic part wasconcentrated by rotary evaporation. The reaction mixture was finallypurified by silica gel column chromatography (Hex:EtOAc/7:3 to 1:1), seeFIG. 8. The3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine(2) was obtained as white powder in 75% yield (1.75 g, R_(f)=0.6,hex:EtOAc/1:1). ¹H-NMR (CDCl₃): δ_(H) 8.16 (s, 1H), 7.48 (s, 1H), 6.28(m, 1H), 4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H), 3.78-3.90 (m, 2H),2.39 (m, 1H), 2.14, 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s, 3H), 0.93 (s,9H), and 0.13 (s, 3H) ppm.

Example 2 Synthesis of 3′-O-(ethyldithiomethyl)-2′-deoxythymidine (4)

Compound 2 (1.75 g, 4.08 mmol), dried overnight under high vacuum,dissolved in 20 mL dry CH₂Cl₂ was added with Et₃N (0.54 mL, 3.87 mmol)and 5.0 g molecular sieve-3A, and stirred for 30 min under Aratmosphere. The reaction flask was then placed on an ice-bath to bringthe temperature to sub-zero, and slowly added with 1.8 eq 1M SO₂Cl₂ inCH₂Cl₂ (1.8 mL) and stirred at the same temperature for 1.0 hour. Thenthe ice-bath was removed to bring the flask to room temperature, andadded with a solution of potassium thiotosylate (1.5 g) in 4 mL dry DMFand stirred for 0.5 hour at room temperature.

Then 2 eq EtSH (0.6 mL) was added and stirred additional 40 min. Themixture was then diluted with 50 mL CH₂Cl₂ and filtered through celite-Sin a funnel. The sample was washed with adequate amount of CH₂Cl₂ tomake sure that the product was filtered out. The CH₂Cl₂ extract was thenconcentrated and purified by chromatography on a silica gel column(Hex:EtOAC/1:1 to 1:3, Rt=0.3 in Hex:EtOAc/1:1). The resulting crudeproduct was then treated with 2.2 g of NH₄F in 20 mL MeOH. After 36hours, the reaction was quenched with 20 mL saturated NaHCO₃ andextracted with CH₂Cl₂ by partitioning. The CH₂Cl₂ part was dried overNa₂SO₄ and purified by chromatography (Hex:EtOAc/1:1 to 1:2), see FIG.8. The purified product (4) was obtained as white powder in 18% yield,0.268 g, R_(f)=0.3, Hex:EtOAc/1:2).

¹H NMR in CDCl₃: δ_(H) 11.25 (1H, S), 7.65 (1H, S), 6.1 (1H, m), 5.17(1H, m), 4.80 (2H, S), 4.48 (1H, m), 3.96 (1H, m), 3.60 (2H, m), 3.26(3H, s), 2.80 (2H, m), 2.20 (2H, m) and 1.14 (3H, m) ppm.

Example 3 Synthesis of the triphosphate of3′-O-(ethyldithiomethyl)-2′-deoxythymidine (5)

In a 25 mL flask, compound 4 (0.268 g, 0.769 mmol) was added with protonsponge (210 mg), equipped with rubber septum. The sample was dried underhigh vacuum for overnight. The material was then dissolved in 2.6 mL(MeO)₃PO under argon atmosphere. The flask, equipped with Ar-gas supply,was then placed on an ice-bath, stirred to bring the temperature tosub-zero. Then 1.5 equivalents of POCl₃ was added at once by a syringeand stirred at the same temperature for 2 hour under Argon atmosphere.Then the ice-bath was removed and a mixture consisting oftributylammonium-pyrophosphate (1.6 g) and Bu₃N (1.45 mL) in dry DMF (6mL) was prepared. The entire mixture was added at once and stirred for10 min. The reaction mixture was then diluted with TEAB buffer (30 mL,100 mM) and stirred for additional 3 hours at room temperature. Thecrude product was concentrated by rotary evaporation, and purified byC18 Prep HPLC (method: 0 to 5 min 100% A followed by gradient up to 50%B over 72 min, A=50 mM TEAB and B=acetonitrile). After freeze drying ofthe target fractions, the semi-pure product was further purified by ionexchange HPLC using PL-SAX Prep column (Method: 0 to 5 min 100% A, thengradient up to 70% B over 70 min, where A=15% acetonitrile in water,B=0.85M TEAB buffer in 15% acetonitrile). Final purification was carriedout by C18 Prep HPLC as described above resulting in 25% yield ofcompound 5, see FIG. 8.

Example 4 Synthesis ofN⁴-Benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2′deoxycytidine (7)

The synthesis of 3′-O-(ethyldithiomethyl)-dCTP (10) was achievedaccording to FIG. 9.N⁴-benzoyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine (6) (50.0 g,112.2 mmol) was dissolved in DMSO (210 mL) in a 2 L round bottom flask.It was added sequentially with acetic acid (210 mL) and acetic anhydride(96 mL), and stirred for 48 h at room temperature. During this period oftime, a complete conversion to product was observed by TLC (R_(f)=0.6,EtOAc:hex/10:1 for the product).

The mixture was separated into two equal fractions, and each wastransferred to a 2000 mL beaker and neutralized by slowly addingsaturated K₂CO₃ solution until CO₂ gas evolution was stopped (pH 8). Themixture was then extracted with EtOAc in a separating funnel. Theorganic part was then washed with saturated solution of NaHCO₃ (2×1 L)followed by with distilled water (2×1 L), then the organic part wasdried over Na₂SO₄.

The organic part was then concentrated by rotary evaporation. Theproduct was then purified by silica gel flash-column chromatographyusing puriflash column (Hex:EtOAc/1:4 to 1:9, 3 column runs, on 15 um,HC 300 g puriflash column) to obtainN4-benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2′-deoxycytidine(7) as grey powder in 60% yield (34.0 g, R_(f)=0.6, EtOAc:hex/9:1), seeFIG. 9.

¹H-NMR of compound 7 (CDCl₃): δ_(H) 8.40 (d, J=7.1 Hz, 1H), 7.93 (m,2H), 7.64 (m, 1H), 7.54 (m, 3H), 6.30 (m, 1H), 4.62 & 4.70 (2Xd, J=11.59Hz, 2H), 4.50 (m, 1H), 4.19 (m, 1H), 3.84 & 3.99 (2Xdd, J=11.59 & 2.79Hz, 2H), 2.72 (m, 1H), 2.21 (m, 1H), 2.18 (s, 3H), 0.99 (s, 9H), and0.16 (s, 6H) ppm.

Example 5N⁴-Benzoyl-3′-O-(ethyldithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine(8)

N⁴-Benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2′-deoxycytidine(7) (2.526 g, 5.0 mmol) dissolved in dry CH₂Cl₂ (35 mL) was added withmolecular sieve-3A (10 g). The mixture was stirred for 30 minutes. Itwas then added with Et₃N (5.5 mmol), and stirred for 20 minutes on anice-salt-water bath. It was then added slowly with 1M SO₂Cl₂ in CH₂Cl₂(7.5 mL, 7.5 mmol) using a syringe and stirred at the same temperaturefor 2 hours under N2-atmosphere. Then benzenethiosulfonic acid sodiumsalt (1.6 g, 8.0 mmol) in 8 mL dry DMF was added and stirred for 30minutes at room temperature. Finally, EtSH was added (0.74 mL) andstirred additional 50 minutes at room temperature. The reaction mixturewas filtered through celite-S, and washed the product out with CH₂Cl₂.After concentrating the resulting CH₂CH₂ part, it was purified by flashchromatography using a silica gel column (1:1 to 3:7/Hex:EtOAc) toobtain compound 8 in 54.4% yield (1.5 g), see FIG. 9. ¹H-NMR of compound8 (CDCl₃): δ_(H) 8.40 (m, 1H), 7.95 (m, 2H), 7.64 (m, 1H), 7.54 (m, 3H),6.25 (m, 1H), 4.69 & 4.85 (2Xd, J=11.60 Hz, 2H), 4.50 (m, 1H), 4.21 (m,1H), 3.84 & 3.99 (2Xdd, J=11.59 & 2.79 Hz, 2H), 2.75 (m, 3H), 2.28 (m,1H), 1.26 (m, 3H), 0.95 (s, 9H), and 0.16 (s, 6H) ppm.

Example 6 N⁴-Benzoyl-3′-O-(ethyldithiomethyl)-2′-deoxycytidine (9)

N⁴-Benzoyl-3′-O-(ethyldithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine(8, 1.50 g, 2.72 mmol) was dissolved in 50 mL THF. Then 1M TBAF in THF(3.3 mL) was added at ice-cold temperature under nitrogen atmosphere.The mixture was stirred for 1 hour at room temperature. Then thereaction was quenched by adding 1 mL MeOH, and solvent was removed after10 minutes by rotary evaporation. The product was purified by silica gelflash chromatography using gradient 1:1 to 1:9/Hex:EtOAc to result incompound 9 (0.78 g, 65% yield, Rf=0.6 in 1:9/Hex:EtOAc), see FIG. 9.¹H-NMR of compound 9 (CDCl₃): δ_(H) 8.41 (m, 1H), 8.0 (m, 2H), 7.64 (m,2H), 7.50 (m, 2H), 6.15 (m, 1H), 4.80 & 4.90 (2Xd, J=11.60 Hz, 2H), 4.50(m, 1H), 4.21 (m, 1H), 4.00 & 3.85 (2Xdd, J=11.59 & 2.79 Hz, 2H), 2.80(m, 2H), 2.65 (m, 1H), 2.40 (m, 1H), and 1.3 (s, 3H) ppm.

Finally, the synthesis of compound 10 was achieved from compound 9following the standard synthetic protocol described in the synthesis ofcompound 5 (see FIG. 8).

Example 7

The synthesis of the labeled nucleotides can be achieved following thesynthetic routes shown in FIG. 10 and FIG. 11. FIG. 10 is specific forthe synthesis of labeled dT intermediate, and other analogs could besynthesized similarly.

Synthesis of5′-O-(tert-butyldimethylsilyl)-5-(N-trifluoroacetyl-aminopropargyl)-2′-deoxyuridine(12)

5′-O-(tert-butyldimethylsilyl)-5-iodo-2′-deoxyuridine (11, 25.0 g, 53.4mmol) was dissolved in dry DMF (200 mL) in a 2-neck-round bottom flask.The reaction flask is flushed with Ar-gas filled balloon. It was thenadded with, freshly opened, vacuum driedtetrakis(triphenylphosphine)palladium (0) (6.16 g, 5.27 mmol) and CuI(2.316 g, 12.16 mmol) and stirred at room temperature for 10 minutesunder argon atmosphere. Next, N-trifluoroacetyl-propargylamine (23.99 g,157.8 mmol, 2.9 eq) and Et₃N (14.7 mL, 105.5 mmol) were addedsequentially. The mixture was stirred for 3.0 hours at room temperatureand reaction completion was confirmed by TLC (R_(f)=0.5 in EtOAc:Hex/3:2for the product).

Solvent was then removed by rotary evaporation. The resulting crudeproduct was dissolved in 500 mL EtOAc and transferred into a separatingfunnel. The organic part was then washed with saturated NaHCO₃ (2×400mL) and saturated NaCl (2×400 mL) solutions, respectively. The EtOAcpart was then dried over anhydrous Na₂SO₄. After filtering off theNa₂SO₄ salt, the filtrate was concentrated using a rotary evaporator. Itwas then purified by a silica gel flash chromatography (1:1 Hex:EtOAc to2:3 Hex:EtOAc, 200 gm, 15 um HP puriflash column, 3 column runs) afterbinding to 3×40 gm silica gel resulting in 21.994 g of 12 (83.88%yield), see FIG. 10.

¹H-NMR in compound 12 (DMF-d₇): δH 11.65 (brs, 1H), 10.15 (brs, 1H),8.15 (brs, 1H, H6), 6.37 (t, J=5.99 Hz, 1H, H1′), 5.42 (m, 1H), 4.41 (m,1H), 4.37 (brs, 2H, for NH—CH₂ of propargylamine group), 4.00 (m, 1H),3.84-3.97 (m, 2H), 2.30 (m, 1H, H2′), 2.20 (m, 1H, H2′), 0.97 (s, 9H,3X—CH₃, TBDMS) and 0.19 (s, 6H, 2×CH₃, TBDMS) ppm.

Example 8 Synthesis of5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-5-(N-trifluoroacetyl-aminopropargyl)-2′-deoxyuridine(13)

Compound 12 (21.99 g, 44.77 mmol) was dissolved in DMSO (90 mL) in a1000 mL round bottom flask. It was then added sequentially with AcOH (40mL) and acetic anhydride (132 mL) and stirred for 48 hours at roomtemperature. The reaction completion was confirmed by TLC (R_(f)=0.5;Hex:EtOAc/1:1 for the product).

The reaction mixture was then transferred to 2,000 mL beaker, andneutralized by saturated K₂CO₃ until the evolution of CO₂ gas was ceased(˜pH 8.0). The mixture was then transferred into a separating funnel andextracted (2×500 mL CH₂Cl₂). The combined organic part was then washedwith saturated NaHCO₃ (1×500 mL) and dried over Na₂SO₄. After filteringoff the Na₂SO₄, the organic part was concentrated by rotary evaporationand purified by silica gel flash chromatography (Hex:EtOAc/7:3 to 1:1)producing 12.38 g of compound 13 (˜50% yield), see FIG. 10. TLC:R_(f)=0.5; Hex:EtOAc/1:1, ¹H-NMR of compound 13 (DMSO-d): δ_(H) 11.69(s, 1H), 10.01 (s, 1H), 7.93 (s, 1H, H6), 6.07 (m, 1H, H1′), 4.69 (m,2H), 4.38 (m, 1H), 4.19 (m, 2H), 4.03 (m, 1H), 3.75 (m, 2H), 2.34 (m,1H), 2.14 (m, 1H), 2.07 (s, 3H), 0.86 (s, 9H) and 0.08 (s, 6H) ppm.

The synthesis of the compounds 14, 15 and 16 can achieved following thesynthetic protocols of the related steps described for compounds 5 and10. Synthesis of other N-trifluoroacetyl-aminopropargyl nucleobases bydescribed as in U.S. Pat. No. 8,017,338 [38], incorporated herein byreference. Removal of the N-trifluoroacetyl group to produce theaminopropargyl nucleobases may be produced by solvolysis under mildconditions [45].

On the other hand, the cleavable linker synthesis can be achieved asshown in FIG. 11, starting from an 1,4-dutanediol and is described inExample 9.

Example 9 Synthesis of4-O-(tert-butyldiphenylsilyl)-butane-1-O-(methylthiomethyl), 18

18.3 g 1,4-butanediol, 17 (18.3 g, 203.13 mmol) dissolved in 100 mL drypyridine in a 1 L flask was brought to sub-zero temperature on anice-bath under nitrogen atmosphere. It was added withtert-butyldiphenylsilylchloride (TBDPSCl, 19.34 g, 70.4 mmol) slowlywith a syringe. The reaction flask was allowed to warm up to roomtemperature with the removal of the ice-bath and stirring continued forovernight at room temperature. The solvent was then removed by rotaryevaporation and purified by flash chromatography using silica gel column(7:3 to 1:1/Hex:EtOAc) resulting in4-O-(tert-butyldiphenylsilyl)-butane-1-ol (13.7 g, 59.5% yield,R_(f)=0.7 with 1:1/Hex:EtOAc, ¹H NMR (CDCl₃): δ_(H) 7.70 (4H, m), 7.40(4H, m), 3.75 (2H, m), 3.65 (m, 2H), 3.70 (4H, m) and 1.09 (9H, m) ppm.Of the resulting product, 6.07 g (18.5 mmol) was dissolved in 90 mL dryDMSO, see FIG. 11. It was then added with acetic acid (15 mL) and aceticanhydride (50 mL). The mixture was stirred for 20 hours at roomtemperature. It was then transferred to a separating funnel and washedwith 300 mL distilled water by partitioning with the same volume ofEtOAc. The EtOAc part was then transferred to a 1,000 mL beaker andneutralized with saturated K₂CO₃ solution. The aqueous part was removedby partitioning and the EtOAc part was then further washed withdistilled water (3×300 mL) and dried over MgSO₄. The EtOAc part was thenconcentrated and purified by flash chromatography on a silica gel column(Hex:EtOAc/97:3 to 90:10) to obtain4-O-(tert-butyldiphenylsilyl)-1-O-(methylthiomethyl)-butane, 18 (5.15 g,71.7% yield, R_(f)=0.8 in 9:1/Hex:EtOAc). ¹H NMR (CDCl₃): δH 7.70 (4H,m), 7.40 (6H, m), 4.62 (2H, s), 3.70 (2H, m), 3.50 (2H, m), 2.15 (2H,s), 1.70 (4H, m) and 1.08 (9H, m) ppm.

Example 10 Synthesis of Compound 19

Compound 18 (2.0 g, 5.15 mmol) was dissolved in 40 mL dry CH₂Cl₂, andadded with 10 g molecular sieve-3A and 0.78 mL Et₃N (5.66 mmol). Themixture was stirred under N2 gas at room temperature for 30 min. Thenthe flask was placed on an ice-bath to bring the temperature tosub-zero. It was then added slowly with 7.7 mL of 1M SO₂Cl₂/CH₂Cl₂solution (7.7 mmol) and stirred under N₂ for 1 hour. Then the ice-bathwas removed and benzenethiosulfonic acid-Na salt (1.6 g, 8.24 mmol) in 8mL DMF was added and stirred for 30 minutes at room temperature. Then4-mercaptophenylacetic acid (1.73 g, 10.3 mmol, 2.0 eq) in 7 mL dry DMFwas added and stirred for 2 hours. The entire crude sample was thenfiltered through celite-S and the product was washed out by EtOAc. EtOAcextract was then concentrated by rotary evaporation and purified on asilica gel column (1:1 to 3:7/Hex:EtOAc) to obtain 1.19 g of compound 19in 43% yield, see FIG. 11, R_(f)=0.5 Hex:EtOAc/3:7. ¹H NMR (CDCl₃): 7.65(4H, m), 7.55 (2H, m), 7.45 (6H, m), 7.20 (2H, s), 4.80 (2H, m), 3.65(4H, m), 3.50 (2H, m), 1.60 (4H, m), and 1.09 (9H, s) ppm.

Example 11 Synthesis of Compound 20

Compound 19 (0.6 g, 1.11 mmol) dissolved in 20 mL dry DMF was treatedwith DSC (0.426 g, 1.5 eq) and Et₃N (0.23 mL) at room temperature andstirred for 1.5 hours under nitrogen atmosphere. Then a mixtureconsisting of 11-azido-3,6,9-trioxadecan-1-amine (2.0 eq) and Et₃N (2.0eq) was prepared in 5 mL DMF. The entire solution was added to thereaction mixture at once and stirred for 1 hour. The solvent was thenremoved under vacuum and purified by silica gel flash chromatographyusing gradient 0 to 10% CH₂Cl₂:MeOH to obtain compound 20 in 36% yield(0.297 g, R_(f)=0.8, 10% MeOH:CH₂Cl₂), see FIG. 11. ¹H NMR (MeOH-4):δ_(H) 7.70 (4H, m), 7.55 (2H, m), 7.40 (6H, m), 7.45 (2H, m), 4.85 (2H,s), 3.65-3.30 (22H, m), 1.65 (4H, m), and 1.09 (9H, m) ppm.

Then, the product 20 (0.297 g) was dissolved in 7 mL dry THF in a flaskand placed on an ice-bath to bring to sub-zero temperature undernitrogen atmosphere. Then 0.6 mL 1M TBAF in THE was added drop-wise andstirred for 3 hours at ice-cold temperature. The mixture was quenchedwith 1 mL MeOH and volatiles were removed by rotary evaporation andpurified by flash chromatography to obtain 165 mg of the product 21, seeFIG. 11, ¹H NMR (MeOH-d4): δH 7.55 (2H, m), 7.25 (2H, m), 4.85 (2H, s),3.75-3.30 (22H, m) and 1.50 (4H, m) ppm. This product can be coupled toalkyne substituted dye using click chemistry and to nucleotide using CDIas activating agent to result in compound 22.

Another variant of cleavable linker, where the stabilizing gem-dimethylgroup attached to α-carbon of the cleavable linker, can be achievedfollowing FIG. 12.

Example 12

In another aspect, the cleavable linker can be compound 30, where thedisulfide is flanked by gem-dimethyl groups and attached to a flexibleethylene glycol linker (PEG). The linker is attached to thePA-nucleotide (e.g. compound 33) via carbamate group (—NH—C(═O)O—). Theresulting nucleotide analogue in such case can be as in compound 35(dUTP analogue), which can be synthesized according to the FIG. 13.Other nucleotide analogues (e.g. analogues of dATP, dGTP, dCTP) can besynthesized similarly by replacing PA-nucleotide 33 with appropriatePA-nucleotide analogues at the last step of the reaction sequence.

Example 13 Synthesis of Compound 28

Compound 18 (15.53 g, 40 mmol) (see Example 9) for synthesis of compound18) was dissolved in 450 mL of dry dichloromethane in a round bottomflask. Molecular sieves (3 Å, 80 g) and triethylamine (5.6 mL) wereadded, and the reaction mixture was stirred at 0° C. for 0.5 hour undernitrogen atmosphere. Next, SO₂Cl₂ (1 M in DCM, 64 mL) was added slowlyby a syringe and stirred for 1.0 hour at 0° C. temperature. Then,ice-water bath was removed, and a solution of potassium-thiotosylate(10.9 g, 48.1 mmol) in 20 mL anhydrous DMF was added at once and stirredfor 20 minutes at room temperature. The reaction mixture was then pouredinto 3-mercapto-3-methylbutan-1-ol (4.4 mL, 36 mmol) dissolved in 20 mLDMF in a 2 L round-bottom flask. The resulting mixture was stirred for0.5 hours at room temperature, and filtered through celite. The productwas extracted with ethyl acetate. The combined organic extracts werewashed with distilled water in a separatory funnel, followed byconcentrating the crude product by rotary evaporation. The product (28)was obtained in 26% yield (5.6 g) after purification by flashchromatography on silica gel using EtOAc:Hexane as mobile phase, seeFIG. 13. ¹H NMR (CDCl₃): δ_(H) 7.67-7.70 (m, 4H), 7.37-7.47 (m, 6H),4.81 (s, 2H), 3.81 (t, J=6.73 Hz, 2H), 3.70 (t, J=6.21 Hz, 2H), 3.59 (t,J=6.55, 2H), 1.90 (t, J=6.95 Hz, 2H), 1.58-1.77 (m, 4H), 1.34 (s, 6H),and 1.07 (s, 9H) ppm.

Example 14 Synthesis of Compound 29

Compound 28 (5.1 g, 10.36 mmol) was dissolved in 100 mL anhydrouspyridine in a 500 mL round bottom flask. To this solution,1,1′-carbonyldiimidazole (CDI) (3.36 g, 20.7 mmol) was added in oneportion and the reaction was stirred for 1.0 hour at room temperatureunder a nitrogen atmosphere. Then, the reaction mixture was poured intoa solution consisting of 2,2′-(ethylenedioxy)bis(ethylamine) (7.6 mL,51.8 mmol) and anhydrous pyridine (50 mL). The mixture was stirred for1.0 hour at room temperature, and the volatiles were removed by rotaryevaporation. The resulting crude product was purified by flashchromatography on silica using MeOH:CH₂Cl₂/9.5:0.5 to furnish purecompound 29 (4.4 g, 65% yield), see FIG. 13. ¹H NMR (CDCl₃): δ_(H)7.63-7.68 (m, 4H), 7.34-7.44 (m, 6H), 4.76 (s, 2H), 4.17 (t, J=7.07 Hz,2H), 3.65 (t, J=6.16 Hz, 2H), 3.60 (s, 4H), 3.49-3.51 (m, 6H), 3.31-3.39(m, 2H), 2.88 (m, 2H), 1.9 (t, J=7.06 Hz, 2H), 1.57-1.73 (m, 4H), 1.31(s, 6H) and 1.03 (s, 9H) ppm.

Example 15 Synthesis of Compound 31

Compound 29 (0.94 g, 1.42 mmol) was dissolved in 40 mL dry THF andtreated with 1M TBAF in THF (1.6 mL, 1.6 mmol) at 0° C. under nitrogenatmosphere. The reaction mixture was stirred for 2.0 hours at 0° C.,during which time LC-MS confirmed complete removal of the TBDPSprotecting group. After removing solvent by rotary evaporation, theproduct was purified by flash chromatography on C18 Flash Column(gradient: 0-100% B over 50 minutes, where A=50 mM TEAB andB=acetonitrile). The target fractions were combined and lyophilizedresulting in pure compound 30 (0.284 g, 47% yield), MS (ES+) calculatedfor (M+H) 429.21, observed m/z 429.18. Next, compound 30 (0.217 g, 0.51mmol) was dissolved in 13 mL of dry acetonitrile under a nitrogenatmosphere. To this solution, DIPEA (97.7 uL, 0.56 mmol) and Fmoc-NHSester (273.6 mg, 0.81 mmol) were added at 0° C. temperature and stirredfor 2.0 hours at the same temperature. The product was then purified byflash chromatography on silica gel, 1:1 to 1:9/hex:EtOAc gradient,leading to a semi-pure product, which was further purified using2-5%/MeOH—CH₂Cl₂ gradient to obtain compound 31 (0.245 g, 74% yield),see FIG. 13. ¹H NMR (CDCl₃): δ_(H) 7.70 (2H, d, J=7.3 Hz), 7.59 (2H, d,J=7.6 Hz), 7.32 (2H, m), 7.24 (2H, m), 4.69 (2H, s), 4.35 (2H, m), 4.16(1H, m), 4.09 (2H, m), 3.60-3.45 (12H, m), 3.36-3.26 (4H, m), 1.82 (2H,m), 1.60 (4H, m) and 1.22 (6H, s) ppm.

Example 16 Synthesis of Compound 32

Compound 31 (93 mg, 0.143 mmol) was dissolved in dry acetonitrile (12.0mL) in a round bottom flask equipped with magnetic bar and a nitrogengas source. To this solution, DSC (56 mg, 0.21 mmol) and DIPEA (37.4 μL,0.21 mmol) were added sequentially, and the resulting mixture wasstirred at room temperature for 5.0 hours. Additional DSC (48 mg, 0.18mmol) and DIPEA (37.4 μL, 0.21 mmol) were added and stirring continuedfor 15.0 hours at room temperature, during which time TLC showed fullconversion to the activated NHS ester. The product 32 was obtained (59mg, 53% yield) as a thick oil following silica gel flash chromatographypurifications using hexane-ethyl acetate (3:7 to 1:9) gradient and wasused in the next step, see FIG. 13. ¹H NMR (CDCl₃): δ_(H) 7.70 (2H, d,J=7.53 Hz), 7.53 (2H, d, J=7.3 Hz), 7.33 (2H, m), 7.24 (2H, m), 4.69(2H, s), 4.34 (2H, m), 4.28 (2H, m), 4.16 (1H, m), 4.09 (2H, m),3.57-3.46 (10H, m), 3.35-3.26 (4H, m), 2.75 (4H, s), 1.74 (4H, m), 1.62(2H, m) and 1.23 (6H, s) ppm.

Example 17 Synthesis of Compound 34

An aliquot of compound 33 (10 μmols) (synthesized according to Ref. US2013/0137091 A1) was lyophilized to dryness in a 15 mL centrifuge tube.It was then re-suspended in 1.0 mL of dry DMF with 60 μmols DIPEA. In aseparate tube, compound 32 (30 μmols, 3 eq) was dissolved in 3.33 mL dryDMF, and added all at once. The reaction was mixed well by rigorousshaking by hand and placed on the shaker for 12 h at room temperature.Next, piperidine (0.33 mL) was added and shaking continued for 30minutes at room temperature. The product was then purified by HPLC usingC18 column (gradient: 0-70% B over 40 minutes, where A=50 mM TEAB andB=acetonitrile). The product 34 was obtained in 73.3% yield (7.33 umols)after lyophilization of the target fractions, see FIG. 13.

Example 18 Synthesis of Compound 35

An aliquot of compound 34 (4.9 μmols) was dissolved in 1.0 mL distilledwater and 0.5M Na₂HPO₄ (0.49 mL) in a 15 mL centrifuge tube. In aseparate tube, 10 mg of 5-CR₆G-NHS ester (17.9 μmol) was dissolved in0.9 mL of dry DMF. This solution was then added to the reaction mixtureall at once and stirred at room temperature for 6.0 hours. The reactionmixture was then diluted with 50 mM TEAB (25 mL). The product waspurified by HPLC C18 (gradient: 0-60% B over 70 minutes). Compound 35was obtained after lyophilization of the target fractions (2.15 μmol,44% yield in ˜98% purity by HPLC, and the structure was confirmed by MS(ES+): calculated for (M−H) C₅₈H₇₆N₁₀O₂₅P₃S2⁻, 1469.36, found m/z1469.67, see FIG. 13.

Similarly, analogs of dATP, dCTP and dGTP were synthesized followingsimilar procedure described for compound 35, and characterized by HPLCand LC-MS resulting a full set of A-series (98, 100, 101, and 102, FIG.45). For dATP analog calculated for (M−H) C₆₆H₈₃N₁₂O₂₃P₃S₂, 1,568.4348,found m/z 1,568.4400; For dCTP analog calculated for (M−H)C₅₂H₆₅N₁₁O₃₀P₃S₄, 1,545.2070, found m/z 1,545.2080 and for dGTP analogcalculated for (M−H) C₆₆H₉₃N₁₂O₂₇P₃S₄, 1,706.4369, found m/z 1,706.4400.In another aspect, the invention involves nucleotides with cleavablelinker as in compound 43 for dATP analogue where the cleavable disulfideis flanked by gem-dimethyl group and the linker is attached toPA-nucleotide via urea group (—NH(C═O)NH—). The compound can besynthesized according to FIG. 14 (for dATP analogue). For othernucleotide analogues (e.g. for analogues of dCTP, dGTP, dUTP) can besynthesized similarly replacing 42 by appropriate PA-analogues at thelast step of the reaction sequence.

Example 19 Synthesis of Compound 37

In a 1 L round bottom flask with equipped with stir bar,5-(fmoc-amino)-1-pentanol (36, 20 g, 62 mmol) was dissolved in DMSO (256mL) at room temperature. To the solution, AcOH (43 mL) and Ac₂O (145 mL)were added sequentially. The flask was closed with a rubber septum,placed under N2, and stirred at room temperature for 20 h. Reactioncompletion was confirmed by TLC. The reaction mixture was thentransferred to a 3 L beaker and the flask was washed with water. Thebeaker was cooled in an ice bath and the reaction mixture wasneutralized with 50% saturated K₂CO₃ (400 mL) for 30 minutes. Themixture was transferred to a separatory funnel and extracted with EtOAc(2×700 mL). The organic phase was then washed with 50% saturated K₂CO₃(2×400 mL), dried over Na₂SO₄, filtered and concentrated in vacuo. Thecrude oil was purified by silica gel chromatography (0 to 20% B over 20min, A=Hex, B=EtOAc). Collection and concentration of fractions yieldscompound 37 (17.77 g, 75%) as a white solid, see FIG. 14. ¹H NMR(CDCl₃): δH 7.79 (d, J=7.33, 2H), 7.63 (d, J=7.83, 2H), 7.441 (t,J=7.33, 2H), 7.357 (t, J=7.58, 2H), 4.803 (bs, 1H), 4.643 (s, 2H), 4.43(d, J=6.82, 2H), 4.24 (t, J=6.82, 1H), 3.54 (t, J=6.32, 2H), 3.251 (m,1H), 2.167 (s, 3H), 1.657-1.550 (m, 4H), and 1.446-1.441 (m, 2H) ppm.

Example 20 Synthesis of Compound 38

Compound 37 (2.77 g, 7.2 mmol) was dissolved in DCM (60 mL) in a 250 mLround bottom flask equipped with stir bar and septum under N2. To theflask, triethylamine (3.0 mL, 21.6 mL, 3 eq) and 4 Å Molecular Sieves(28 g) were added. The suspension was stirred for 10 min at roomtemperature, followed by 30 min in an ice bath. To the flask was addedSO₂Cl₂ (M solution in DCM, 14.4 mL, 14.4 mmol, 2 eq) and the reactionmixture was stirred in the ice bath for 1 h. Reaction progress wasmonitored by the disappearance of starting material via TLC (1:1Hex:EtOAc). Once SO₂Cl₂ activation was complete, a solution of potassiumthiotosylate (2.45 g, 10.8 mmol, 1.5 eq) in DMF (60 mL) was rapidlyadded. The reaction mixture was allowed to slowly warm to roomtemperature for 1 h. The flask was then charged with3-mercapto-3-methylbutanol (1.8 mL, 14.4 mmol, 2 eq) and stirred at roomtemperature for 1 h. The reaction mixture was filtered and concentratedin vacuo at 40° C. Purification by FCC (0 to 50% B over 30 min, A=Hex,B=EtOAc) afforded 38 (482 mg, 14%) as a yellow oil, see FIG. 14. ¹H NMR(CDCl₃): δ_(H) 7.76 (d, J=7.81, 2H), 7.59 (d, J=7.32, 2H), 7.40 (t,J=7.32, 2H), 7.31 (t, J=7.32, 2H), 4.87 (bs, 1H), 4.79 (s, 2H), 4.40 (d,J=6.84, 2H), 4.21 (t, J=6.84 1H), 3.78 (t, J=6.84, 2H), 3.57 (t, J=6.35,2H), 3.20 (m, 2H), 1.88 (t, J=6.84, 2H), 1.64-1.50 (m, 4H), 1.42-1.39(m, 2H) and 1.32 (s, 6H) ppm.

Example 21 Synthesis of Compound 39

Compound 38 (135 mg, 0.275 mmol) was desiccated under vacuum for 2 h ina 50 mL round bottom flask. The vacuum was removed and the flask placedunder N2. Compound 38 was dissolved in DMF (3.1 mL) and the flask wascharged with DIPEA (96 μL, 0.55 mmol, 2 eq). The solution was stirredfor 10 min and then DSC (120 mg, 0.468 mmol, 1.7 eq) was added in onedose as a solid. The reaction mixture was allowed to stir for 2 h andcompletion was verified via TLC (1:1 Hex:EtOAc). The reaction was thenconcentrated in vacuo at 35° C. and further dried under high vacuum for1 h. The crude oil was loaded on to silica gel and purified by FCC (0 to50% B over 14 min, A=hex, B=EtOAc). The fractions were checked by TLCand concentrated to afford compound 39 (133 mg, 76%) as an oil thatcrystallized over time, see FIG. 14. ¹H NMR (CDCl₃): δ_(H) 7.78 (d,J=7.58, 2H), 7.61 (d, J=7.58, 2H), 7.42 (t, J=7.58, 2H), 7.33 (t, J7.58, 2H), 4.87 (bs, 1H), 4.80 (s, 2H), 4.48 (t, J=7.07, 2H), 4.44 (d,J=6.82, 2H), 4.24 (t, J=7.07, 1H), 3.58 (t, J=6.32, 2H), 3.22 (m, 2H),2.83 (s, 4H), 2.08 (m, 2H), 1.649-1.562 (m, 4H), 1.443-1.390 (m, 2H) and1.366 (s, 6H) ppm.

Example 22 Synthesis of Compound 40

2,2′-(Ethylenedioxy)bis(ethylamine) (92 μL, 635 μmol, 10 eq) andtriethylamine (176 μL, 1270 μmol, 20 eq) were dissolved in DMF (10 mL).A separate solution of 6-ROX, NHS ester (40 mg, 64 umol, 1 eq) in DMF(2.7 mL) was also prepared. The 6-ROX, NHS ester solution was addeddrop-wise to a rapidly stirring solution containing the diamine. Thereaction stirred for 2 h and progress was monitored by C18 HPLC-MS (0 to100% B over 10 min, A=50 mM TEAB, B=MCCN). Once complete, the reactionwas purified via preparative C18-HPLC (10 to 100% B over 50 min, A=50 mMTEAB, B=MCCN). The fractions were combined and lyophilized to yieldcompound 40 (20 mg, 48%) as a purple-red solid, see FIG. 14. MS (ES−)calculated for (M−H) C₃₉H₄₅N₄O₆ 664.33, found m/z 664.56.

Example 23 Synthesis of Compound 41

Compound 40 (10 mg, 15 μmol) was dissolved in DMF (1 mL) and chargedwith DIPEA (8 μL, 45 μmol, 3 eq). Separately, compound 39 (28 mg, 45μmol, 3 eq) was dissolved in DMF (0.21 mL). The solution of compound 39was rapidly added to the solution with compound 40. The reaction wasplaced on a shaker plate for 1.5 h at which time analytical C18-HPLC(0-100% B over 10 min, A=50 mM Acetate Buffer pH 5.2, B=MCCN) revealedremaining compound 40. Additional compound 39 (13 mg, 21 μmol, 1.4 eq)was added and the reaction was placed on a shaker plate for anadditional hour. Without additional analytics, piperidine (300 μL) wasadded and allowed to react for 10 min. The reaction mixture was thendirectly injected on to a preparative C18-HPLC (10-100% B over 50 min,A=50 mM TEAB, B=MeCN). The fractions were collected and lyophilized toyield compound 41 (4.7 mg, 34%) as a purple-red solid, see FIG. 14. MS(ES+) calculated for (M+H) C₅₁H₆₈N₅O₉S₂ ⁺ 959.45, found m/z 959.76.

Example 24 Synthesis of Compound 43

A 5 mL sample vial was charged with amine 41 (2 mg, 2 μmol), DSC (0.8mg, 3 μmol, 1.5 eq), DIPEA (0.7 μL, 4 μmol, 2 eq), andN,N-dimethylformamide (1.7 mL). The reaction mixture was placed on ashaker for 1 h. Reaction progress was monitored by C18-HPLC (0 to 100% Bover 10 min, A=50 mM Acetate Buffer pH 5.2, B=MeCN). Next, nucleotide 42(6 umol, 3 eq, Ref. US 2013/0137091 A1) in 0.1 Na₂HPO₄ (3.3 mL) wasadded and the reaction mixture was placed on a shaker overnight. Thereaction was next diluted with water and purified by preparativeC18-HPLC (0 to 60% B over 70 min, A=50 mM TEAB, B=MeCN) to give thetitle compound 43 (0.5 μmol, 25%), see FIG. 14. MS (ES−) calculated for(M−H) C₆₇H₈₇N₁₃O₂₂P₃S₂ 1581.47, found m/z 1581.65.

Example 25

In another aspect, the cleavable linker can be compound 45, where thelinker is tethered to PA-nucleotides via urea functionality and thedisulfide is connected to the dye by a two carbon linker. The resultingnucleotide analogue in such case can be as in compound 49 (dGTPanalogue), which can be synthesized according to the FIG. 15. Othernucleotide analogues (e.g. analogues of dATP, dUTP, dCTP) can besynthesized similarly by replacing nucleotide 46 with appropriatePA-nucleotide analogues in the third step of the reaction sequence.

Example 26 Synthesis of Compound 44

A 100 mL round bottomed flask equipped with a magnetic stir bar wascharged with 37 (1.00 g, 2.59 mmol) in CH₂Cl₂, molecular sieves andtriethylamine (0.72 mL, 5.18 mmol). The reaction mixture was stirred for10 minutes at room temperature and cooled to 0° C. Sulfuryl chloride(4.40 mL, 4.40 mmol) was added slowly and the resultant mixture wasstirred for 1 hour at 0° C. TLC analysis using 20% ethyl acetate inhexanes indicated the disappearance of starting material, and a solutionof benzenethionosulfonic acid sodium salt (648 mg, 3.89 mmol) inN′,N′-dimethylformamide (5 mL) was added in one portion at 0° C. and thereaction mixture was stirred for 20 min at room temperature. Next,N-(trifluoroacetamido)ethanethiol (896 mg, 5.18 mmol) was added in oneportion and the resulting mixture was stirred for 30 minutes at roomtemperature. The molecular sieves were filtered off and the solventswere removed under reduced pressure and the residue was purified viacolumn chromatography on silica gel using 0-20% ethyl acetate-hexanesgradient, to give the title compound 44 (529 mg, 39%) as a yellowishoil. ¹H NMR (CDCl₃), see FIG. 15: δ_(H) 7.76 (d, J=7.52 Hz, 2H), 7.57(d, J=7.50 Hz, 2H), 7.40-7.38 (m, 2H), 7.30-7.25 (m, 2H), 4.82 (s, 2H),4.42 (d, 2H), 4.21-4.20 (m, 1H), 3.70-3.67 (m, 2H), 3.59-3.55 (m, 2H),3.17-3.16 (m, 2H) and 1.64-1.40 (m, 6H) ppm.

Example 27 Synthesis of Compound 45

A 25 mL round bottomed flask equipped with a magnetic stir bar wascharged with carbamate 44 (100 mg, 0.184 mmol), and 1 mL of 20%piperidine solution in N,N-dimethylformamide at room temperature. Thereaction mixture was stirred at room temperature for 10 minutes, thendiluted with acetonitrile (5 mL) and purified via reverse phasepreparative HPLC using a 0-30% acetonitrile-TEAB buffer gradient to givethe title compound 45 (11 mg, 20%) as a clear oil, see FIG. 15. ¹H NMR(400 MHz, CD₃OD) δ_(H) 4.90 (s, 2H), 3.64-3.60 (m, 2H), 3.32 (s, 2H),2.98-2.93 (m, 2H), 2.86-2.82 (m, 2H), 1.66-1.60 (m, 2H), 1.50-1.48 (m,2H) and 1.33-1.30 (m, 2H) ppm.

Example 28 Synthesis of Compound 47

A 5 mL sample vial was charged with amine 45 (0.960 mg, 3.0 μmol), DSC(1.15 mg, 4.5 μmol) and triethylamine (60 μL, 6.0 μmol) and shaken for 2hours at room temperature. Then a solution consisting of 3 eq ofnucleotide 46 in 200 μL (ref. US 2013/0137091 A1) inN,N-dimethylformamide was added. The reaction mixture was placed on ashaker for 12 hours. The reaction was next diluted with TEAB buffer andpurified by preparative reverse phase HPLC using a 0-30% acetonitrile:50 mM TEAB buffer gradient to give the title compound 47 (in 14% yield),see FIG. 15. MS (ES−): calculated for (M−H) C₂₆H₃₇F₃N₁₀O₁₆P₃S₂ ⁻,959.10, found m/z 959.24.

Example 29 Synthesis of Compound 48

Nucleotide 47 (1 μmol) was dissolved in TEAB buffer (200 μL of 50 mMaqueous soln.) and treated with 200 μL of ammonium hydroxide (30%aqueous soln.) for 50 minutes at room temperature. The reaction was thendiluted with TEAB buffer (1 mL of 1M solution) and distilled water (5mL). The resulting mixture was purified via C18-HPLC, 0-30%Acetonitrile: 50 mM TEAB buffer gradient to afford the title compound 48(0.40 μmol, 90%), see FIG. 15. MS (ES−): calculated for (M−H)C₂₄H₃₈N₁₀O₁₅P₃S₂ ⁻,863.12, found m/z 863.45.

Example 30 Synthesis of Compound 49

An aliquot of compound 48 (0.04 μmols) was dissolved in 0.1 mL distilledwater and 0.5M Na₂HPO₄ (20 μL) in a 3 mL eppendorf tube. In a separatetube, 1 mg of ROX-NHS ester (0.168 μmol) was dissolved in 48 μL of dryDMF. This solution was then added to the reaction mixture all at onceand stirred at room temperature for 6.0 hours. The reaction mixture wasthen diluted with 50 mM TEAB (5 mL). The product was purified byC18-HPLC using (0-60% B gradient, A=50 mM TEAB, B=acetonitrile).Compound 49 was obtained after lyophilization of the target fractions(0.03 μmol, 30% yield), see FIG. 15. MS (ES−) calculated for (M−H),Cs₇H₆₇N₁₂O₁₉P₃S₂ ⁻ 1380.33, found 1380.25.

Cleavage Comparison with Regular Disulfide Linked Nucleotides

This new class of nucleotides containing cleavable oxymethylenedisulfide(—OCH₂—SS—) linker, disclosed herein, was compared with regulardisulfide (—SS—) linked nucleotide (e.g. nucleotide 50, described in USPat. Appln. 2013/0137091 [46]) under reducing phosphine based cleavageconditions. A stark difference in these two classes of nucleotides wasobserved. When labeled nucleotide 50 was exposed to 10 eq of TCEP at 65°C., it generated a number of side products including compound 52 alongwith the expected product 51 identified by LC-MS (FIG. 16, and FIG. 17,5 minutes exposure). The proportion of the unwanted side productsincreased over time (FIG. 18, 15 minutes exposure). Under identicalcleavage conditions, the oxymethylenedisulfide linked nucleotide 35cleanly produced the desired cleavage products, compounds 53 and 54. Themethylene thiol segment (—CH₂SH) of the linker was fully eliminated fromthe nucleotide upon cleavage of the disulfide group (FIG. 20 and FIG.21, 5 minutes exposure). In addition, a prolonged exposure to TCEP didnot generate further side products as revealed by LC-MS (FIG. 22, 15minutes exposure). Therefore, this new class of nucleotides could offersignificant advantages in the use of DNA sequencing by synthesis (SBS)by eliminating side reactions inherent to the presence of a thiol groupas shown in FIG. 4.

Example 31 Synthesis of Compound 57

In another embodiment, the 3′—OH group of the nucleotides can be cappedwith —CH₂—SS-Et or —CH₂—SS-Me, and the fluorophore dyes are attached tothe nucleobases via one of the cleavable —OCH₂—SS— linkers describedearlier (e.g. as in compound 35, 43, and 49).

The synthesis of PA nucleotides with 3′-OCH₂—SS-Et and —OCH₂—SS-Me, canbe achieved according to FIG. 10 and FIG. 22, respectively. Thedifference in the synthesis of 3′-OCH₂—SS-Me analogues from that of3′-OCH₂—SS-Et (FIG. 10) is the replacement of mercaptoethanol (EtSH) bymethanethiol or sodium thiomethoxide at the appropriate step as shown inFIG. 22. The —OCH₂—SS-Me group is the smallest structure among allpossible 3′-O—CH₂SS—R analogues. Therefore, nucleotide analogues with3′-OCH₂—SS-Me capping group should perform better than those of otheranalogues in terms of enzymatic incorporation rates and cleavability byreducing agents such as TCEP.

Next, the resultant PA-nucleotide (e.g. 57) can be coupled to theappropriate cleavable —OCH₂—SS— linkers, and finally to fluorophore dyeas shown in the FIG. 23 using the activated linker 32. And othernucleotides with differing dyes can be synthesized similarly using theappropriate PA-nucleotides (e.g. PA analogues of dATP, dGTP, dCTP) andNHS activated dyes (Alexa488-NHS, ROX-NHS, Cy5-NHS ester etc.) achievingnucleotide analogues labeled with different fluorophore reportinggroups.

Example 32

Nucleotide analogues with different linker can be achieved following theprotocols described, as shown in in the synthesis of compounds 60 and 61(FIG. 24).

Diverse sets of 3′—OCH₂—SS-Et and 3′—OCH₂—SS-Me nucleotides withcleavable linkers —OCH₂SS—, but differing in the chain lengths andsubstitution at the α-carbons can be synthesized similarly. Theresulting classes of nucleotides are shown in the FIG. 25, FIG. 26, andFIG. 27. Among nucleotides shown in the FIG. 25, the cleavable linker isflanked by stabilizing gem-dimethyl group attached to flexibleethylene-glycol linker and attached to PA-nucleobase via carbamatefunctional group (—NH—C(C═)—O—), while in FIG. 26, the carbamate groupis replaced by urea group (—NH—C(C═O)—NH—). On the other hand, amongnucleotides shown in FIG. 27, the disulfide group is attached to primarycarbon chain, and tethered to the PA-nucleobase by urea functionalgroup.

Example 33 Synthesis of Compound 64

A 250 mL round bottom flask was charged with compound 62 (3.0 g, 4.58mmol), 25 mL dry CH₂Cl₂, 3-Å molecular sieves (5.0 g) and cyclohexene(0.55 mL, 5.4 mmol). The resulting mixture was stirred for 10 minutes atroom temperature under a nitrogen atmosphere. The reaction flask wasthen placed on an ice-bath and SO₂Cl₂ (6.8 mL, 1M in CH₂Cl₂, 1.5 eq) wasadded slowly via a syringe, and stirred for 1 hour at 0° C. Next, anextra 0.5 eq of SO₂Cl₂ were added to ensure complete conversion tocompound 63. The volatiles were removed under vacuum while keeping thetemperature close to 10° C. The resulting solid was re-suspended in 20mL of dry DMF and kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (2.45 g, 11.44mmol) was dissolved in dry DMF (30 mL) under nitrogen atmosphere, andtreated with NaH (274.5 mg, 60% in oil) producing a grey slurry. Tothis, compound 63 was added at once and stirred at room temperature for3 hrs under nitrogen atmosphere. The reaction mixture was then filteredthrough Celite®-S (20 g) in a funnel eluting the product with EtOAc (100mL). The EtOAc solution was then washed with distilled water (2×100 mL).The EtOAc extract was dried over Na₂SO₄. concentrated by rotaryevaporation, and purified by flash chromatography (column: 120 gRediSepRfOold, gradient: 80% Hex to 50 Hex:EtOAc). See FIG. 43. Thetarget compound (64) was obtained as white solid (1.2 g, 32% yield,R_(f): 0.4, Hex:EtOAc/3:2). ¹H NMR (CDCl₃): δ_(H) 8.13 (m, 3H), 7.43 (m,1H), 7.32 (m, 2H), 6.12 (m, 1H), 6.00 (s, 2H), 4.62 (m, 2H), 4.31 (m,3H), 4.00 (m, 1H), 3.82-3.60 (m, 13H), 2.39 (m, 1H), 1.84 (m, 1H), 0.78(m, 9H), and 0.01 (m, 6H) ppm.

Example 34 Synthesis of Compound 65

Compound 64 (1.2 g 1.46 mmol) was dried under high vacuum with P₂O₅ in adesiccator overnight and dissolved in 30 mL of anhydrous CH₂Cl₂ in a 100mL flask equipped with a magnetic stirrer. To this was addeddimethyldisulfide (0.657 mL, 7.3 mmol), and the reaction flask wasplaced on an ice-bath. Dimethyl(methylthio)sulfonium tetrafluoroborate(DMTSF, 316 mg, 1.1 eq) was then added and stirred for 1.5 hr at 0° C.The reaction mixture was transferred to a 250 mL separatory funnel andneutralized with 50 mL of 0.1M aq. solution of NaHCO₃, and extractedwith CH₂Cl₂ (2×50 mL). See FIG. 43. The organic portion was dried overNa₂SO₄ and concentrated by rotary evaporation. The crude product waspurified on a silica gel column (80 g RediSepRf gold) using gradient80-50% Hex-EtOAc to result in 0.82 g of compound 65 (82% yield,R_(F)=0.5, Hex:EtOAc/3:2). ¹H NMR (CDCl₃): δ_(H) 8.15 (m, 3H), 7.42 (m,1H), 7.35 (m, 2H), 6.11 (m, 1H), 4.80-4.65 (m, 2H), 4.34 (m, 1H), 4.28(m, 2H), 4.10 (m, 1H), 3.83-3.67 (m, 2H), 2.49 (m, 1H), 2.34 (s, 3H),1.90 (m, 1H), 0.78 (m, 9H), and 0.10 (m, 6H) ppm.

Example 35 Synthesis of Compound 66

A round bottomed flask equipped with a magnetic stirrer was charged withcompound 65 (0.309 g, 0.45 mmol) and 10.0 mL dry CH₂Cl₂ (10.0 mL) andplaced on an ice-bath under a nitrogen atmosphere. TBAF (0.72 mL, 0.72mmol, in 1M solution) was added slowly via syringe. The reaction mixturewas stirred for 3 hours at 0° C. The reaction mixture was thentransferred to a separatory funnel and quenched with 0.5 M NaHCO₃solution (50 mL). The resulting mixture was extracted with EtOAc (2×100mL) and dried over Na₂SO₄. The product 66 was obtained as a white powderafter silica gel column chromatography in 76% yield (196 mg, R_(f)=0.3,Hex:EtOAc/1:1) on a 40 g RediSepRf column using gradient 7:3 to 2:3Hex:EtOAc. See FIG. 43. ¹H NMR (CDCl₃): δ_(H) 8.40 (s, 1H), 8.25 (m,2H), 7.60 (m, 1H), 7.52 (m, 2H), 6.21 (m, 1H), 4.90-80 (m, 2H), 4.65 (m,1H), 4.40 (m, 2H), 4.25 (m, 1H), 4.05-3.85 (m, 2H), 2.62 (m, 1H), 2.50(s, 3H) and 2.31 (m, 1H) ppm.

The product 67 was obtained after phosphorylation of compound 66(confirmed by LC-MS m/z (M−H) 611.19 for C₁₄H₂₃N₄O₁₃P₃S₂ for 67) viastandard triphosphate synthesis method (see the synthesis of compound 5for detail and see FIG. 8). It was further converted to dye labeledproducts according to procedure described for compounds presented inFIG. 13, FIG. 14, and FIG. 15.

Example 36 Synthesis of Compound 70

Compound 68 (7.3 g, 13.8 mmol) was dried in a desiccator overnight anddissolved in anhydrous DCM (70 mL) in a dry 500 mL round bottom flaskequipped with a stirbar and rubber septum under an atmosphere of N2.Cyclohexene (1.54 mL, 15.2 mmol, 1.1 equiv) and dry 3-A molecular sieves(16.6 g) were added to the reaction mixture and the resulting suspensionwas stirred for 20 min at 0° C. in an ice-water bath. Next, SO₂Cl₂ (1 Msolution in DCM, 32.7 mL, 2.36 eqiv) was added and the resulting mixturewas stirred at 0° C. for 1 h. Reaction progress was monitored by thedisappearance of the starting material via TLC (100% EtOAc). Once theSO₂Cl₂ activation was complete, a mixture of (MeO)₃BnSH (7.4 g, 34.5mmol, 2.5 eqiv) and NaH (1.32 g, 33.12 mmol, 60% in mineral oil) in DMF(120 mL) was prepared and rapidly added in one portion. The reaction wasallowed to slowly warm to room temperature and stirred for 1 h. Thereaction mixture was filtered and concentrated in vacuo at 40° C.Purification by column chromatography on silica gel (eluted with 0 to60% ethyl acetate:hexanes gradient 15 mins, followed by 60% ethylacetate:hexanes for 45 mins) afforded the desired compound 70 (4.2 g,43.7% yield) as a clear oil. See FIG. 44. ¹H NMR (CDCl₃): δ_(H) 8.72 (s,1H), 8.31 (s, 1H), 7.94 (m, 2H), 7.52 (m, 1H), 7.44 (m, 2H), 6.41 (m,1H), 6.03 (s, 2H), 4.67 (s, 2H), 4.50 (m, 1H), 4.10 (m, 1H), 3.73 (m,13H), 2.52 (m, 2H), 0.81 (s, 9H) and 0.002 (d, 6H) ppm.

Example 37 Synthesis of Compound 71

Compound 70 (2 g, 2.87 mmol) was dissolved in anhydrous DCM (38 mL) in a200 mL round bottom flask equipped with stirbar and a rubber septumunder an atmosphere of N2 and cooled on an ice-water bath. To thismixture was added dimethyldisulfide (1.3 mL, 14.36 mmol, 5 equiv),followed by addition of DMTSF (620 mg, 3.15 mmol, 1.1 equiv) as asolution in DCM (20 mL), in one portion. The resulting mixture wasallowed to slowly warm to room temperature and then stirred for anadditional 4 h. The reaction was quenched by addition of a saturatedaqueous solution of NaHC₃ (100 mL), extracted with DCM (150 mL×2) andEtOAc (200 mL) dried over Na₂SO₄ and concentrated in vacuo. Purificationby column chromatography on silica gel (eluted with 0 to 60% ethylacetate:hexanes gradient 15 mins, followed by 60% ethyl acetate:hexanesfor 45 mins) afforded the desired compound 71 (1 g, 62% yield) as awhite powder. See FIG. 44. ¹H NMR (CDCl₃): δ_(H) 8.69 (s, 1H), 8.24 (s,1H), 7.94 (m, 1H), 7.51 (m, 1H), 7.42 (m, 2H), 6.41 (m, 1H), 4.82 (m,2H), 4.57 (m, 1H), 4.15 (m, 1H), 3.77 (m, 2H), 2.61 (m, 2H), 2.40 (s,3H), 0.81 (s, 9H) and 0.00 (d, 6H) ppm.

Example 38 Synthesis of Compound 72

Compound 71 (562 mg, 1.25 mmol) was dissolved in anhydrous THF (30 mL)in a 100 mL round bottom flask equipped with a stirbar and rubber septumunder an atmosphere of N₂ and cooled on an ice-water bath. TBAF (1.5 mLof 1 M soln. in THF, 1.5 equiv) was then added dropwise and stirred at0° C. for 2 h. The reaction progress was monitored by TLC (100% ethylacetate R_(f) for compound 72=0.205, R_(f) for compound 71=0.627). Uponreaction completion methanol (5 mL) was added, the reaction wasconcentrated on the rotary and the residue was purified via columnchromatography on silica gel (eluted with 0 to 60% ethyl acetate:hexanesgradient 15 mins, followed by 60% ethyl acetate:hexanes for 45 mins) toafford the desired compound 72 (280 mg, 62% yield) as white powder. SeeFIG. 44. ¹H NMR (CDCl₃): δ_(H) 8.69 (s, 1H), 8.02 (s, 1H), 7.95 (m, 2H),7.53 (m, 1H), 7.44 (m, 2H), 6.25 (m, 1H), 4.83 (m, 2H), 4.70 (m, 1H),4.29 (m, 1H), 3.93 (m, 1H), 3.74 (m, 1H), 2.99 (m, 1H), 2.43 (s, 3H) and2.41 (m, 1H) ppm.

Compound 72 was then converted to triphosphate 73 following standardtriphosphate synthesis described earlier (see the synthesis of compound5 in FIG. 8).

Example 39 Synthesis of Compound 108

A 1 L round bottom flask equipped with a stirbar was charged with1,4-butanediol (18.3 g, 203.13 mmol) in 100 mL of anhydrous pyridine andcooled to 0° C. under a nitrogen atmosphere.tert-Butyldiphenylsilylchloride (13.8 mL, 70 mmol) was then addeddropwise via syringe, the reaction was allowed to gradually warm to roomtemperature and stirring continued at rt for 12 h. The volatiles wereremoved by rotary evaporation and the residue absorbed onto 80 grams ofsilica gel. Purification via flash column chromatography on silica gelusing 30 to 50% ethyl acetate in hexanes gradient resulted in4-O-(tert-butyldiphenylsilyl)-butane-1-ol, 108 (13.7 g, 59.5% yield,R_(f)=0.7 with 1:1/hexanes:ethyl acetate, ¹H NMR (CDCl₃): δ_(H) 7.70 (m,4H), 7.40 (m, 6H), 3.75 (m, 2H), 3.65 (2H, m), 1.70 (m, 4H), 1.09 (m,9H,) ppm. The synthesis is illustrated in FIG. 53.

Example 40 Synthesis of Compound 109

A 250 mL round bottom flask equipped with a magnetic stir bar and wascharged with compound 108 (6.07 g, 18.5 mmol) and 90 mL anhydrous DMSO.Acetic acid (15 mL) and acetic anhydride (50 mL) were sequentially addedand the reaction was stirred for 20 h at room temperature, transferredto a separatory funnel and partitioned between 300 mL distilled waterand 300 mL of ethyl acetate. The organic layer was then transferred to a1 L beaker and neutralized using a saturated aqueous K₂CO₃ solution (500mL). The organic layer was washed with distilled water (3×300 mL) anddried over MgSO₄. The volatiles were removed under reduced pressure andthe residue was purified via flash column chromatography on a silica gel(hexanes:ethyl acetate/97:3 to 90:10) to obtain4-O-(tert-butyldiphenylsilyl)-1-O-(methylthiomethyl)-butane, 109 (5.15g, 71.7% yield, R_(f)=0.8 in 9:1/hexanes:ethyl acetate). ¹H NMR (CDCl₃):δ_(H) 7.70 (m, 4H,), 7.40 (m, 6H), 4.62 (s, 2H), 3.70 (m, 2H), 3.50 (m,2H,), 2.15 (s, 2H), 1.70 (m, 4H), 1.08 (m, 9H) ppm. The synthesis isillustrated in FIG. 53.

Example 41 Synthesis of Compound 110

A 1 L round bottom flask equipped with a magnetic stirbar was chargedwith compound 109 (15.5 g, 40 mmol), anhydrous dichloromethane (450 mL),3 Å molecular sieves (80 g) and triethylamine (5.6 mL) and the reactionwas stirred at 0° C. for 30 min under a nitrogen atmosphere. Next,SO₂Cl₂ (64 mL of 1 M soln. in dichloromethane) was added slowly viasyringe and stirred for 1 h at 0° C. Ice bath was then removed and asolution of potassium-thiotosylate (10.9 g, 48.1 mmol) in 20 mLanhydrous DMF was added at once. The resulting mixture was stirred for20 min at room temperature, added at once to a 2 L round bottom flaskcontaining a solution of 3-mercapto-3-methylbutan-1-ol (4.4 mL, 36 mmol)in DMF (20 mL). The reaction was stirred for 30 min at room temperature,and then filtered through celite-S. The product was partitioned betweenequal amounts of ethyl acetate and water. The organic extracts werewashed with distilled water in a separatory funnel, followed byconcentrating the crude product by rotary evaporation. Purification byflash column chromatography on silica gel using ethyl acetate:hexanesgradient gave the title compound 110 (5.6 g, 26%). ¹H NMR (CDCl₃): δ_(H)7.67-7.70 (m, 4H), 7.37-7.47 (m, 6H), 4.81 (s, 2H), 3.81 (t, J=6.73 Hz,2H), 3.70 (t, J=6.21 Hz, 2H), 3.59 (t, J=6.55, 2H), 1.90 (t, J=6.95 Hz,2H), 1.58-1.77 (m, 4H), 1.34 (s, 6H), and 1.07 (s, 9H) ppm. Thesynthesis is illustrated in FIG. 53.

Example 42 Synthesis of Compound 111

A 500 mL round bottom flask equipped with a magnetic stir bar wascharged with compound 110 (5.1 g, 10.36 mmol), anhydrous pyridine (100mL) and 1,1′-carbonyldiimidazole (CDI) (3.36 g, 20.7 mmol) under anitrogen atmosphere. The reaction mixture was stirred for 1 h at roomtemperature and poured into a solution of2,2′-(ethylenedioxy)bis(ethylamine)(7.6 mL, 51.8 mmol) in anhydrouspyridine (50 mL). Stirring continued for 1 h and the volatiles wereremoved by rotary evaporation. The resulting crude was purified viaflash column chromatography on silica gel using (0-15% methanol inCH₂Cl₂) to furnish compound 111 (4.4 g, 65% yield). ¹H NMR (CDCl₃):δ_(H) 7.63-7.68 (m, 4H), 7.34-7.44 (m, 6H), 4.76 (s, 2H), 4.17 (t,J=7.07 Hz, 2H), 3.65 (t, J=6.16 Hz, 2H), 3.60 (s, 4H), 3.49-3.51 (m,6H), 3.31-3.39 (m, 2H), 2.88 (m, 2H), 1.9 (t, J=7.06 Hz, 2H), 1.57-1.73(m, 4H), 1.31 (s, 6H) and 1.03 (s, 9H) ppm. The synthesis is illustratedin FIG. 53.

Example 43 Synthesis of Compound 113

A 50 mL round bottom flask equipped with a magnetic stir bar was chargedwith compound 111 (0.94 g, 1.42 mmol), anhydrous THF (40 mL) and of TBAF(1.6 mL of 1 M soln. in THF, 1.6 mmol) at 0° C. under nitrogenatmosphere. The reaction mixture was stirred for 2.0 h at 0° C., duringwhich time LC-MS showed complete removal of the TBDPS protecting group.After removing the volatiles on the rotary, the product was purified viaflash chromatography on silica gel (0-5% methanol in dichloromethanegradient, to give pure compound 112 (0.284 g, 47% yield), MS (ES+)calculated for (M+H) 429.21, observed m/z 429.18.

Next, compound 112 (0.217 g, 0.51 mmol) was dissolved in anhydrousacetonitrile (13 mL) under a nitrogen atmosphere and cooled to 0° C.DIPEA (97.7 μL, 0.56 mmol) and Fmoc-NHS ester (273.6 mg, 0.81 mmol) wereadded and the reaction stirred at 0° C. for 2 h. Purification by flashcolumn chromatography on silica gel, using 50 to 90% ethyl acetate inhexanes gradient, produced a semi-pure product, which was furtherpurified via column chromatography on silica gel using 2-5% methanol inCH₂Cl₂ gradient to furnish compound 113 (0.245 g, 74% yield). ¹H NMR(CDCl₃): δ_(H) 7.70 (2H, d, J=7.3 Hz), 7.59 (2H, d, J=7.6 Hz), 7.32 (2H,m), 7.24 (2H, m), 4.69 (2H, s), 4.35 (2H, m), 4.16 (1H, m), 4.09 (2H,m), 3.60-3.45 (12H, m), 3.36-3.26 (4H, m), 1.82 (2H, m), 1.60 (4H, m)and 1.22 (6H, s) ppm. The synthesis is illustrated in FIG. 53.

Example 44 Synthesis of Compound 114

A 50 mL round bottom flask equipped with a magnetic stir bar was chargedwith compound 7 (170 mg, 0.26 mmol), anhydrous acetonitrile (15 mL), DSC(100 mg, 0.39 mmol) and DPIEA (68 μL, 0.39 mmol). The reaction mixturewas stirred at room temperature for 3 h and additional DSC (100 mg, 0.39mmol) and DIPEA (68 μL, 0.39 mmol) were added. The resulting mixture wasstirred at room temperature for 12 h. Reaction progress was followed byTLC (R_(f)=0.4 for starting material, product R_(f)=0.8 in 9:1/ethylacetate:hexanes). The volatiles were removed by rotary evaporation, andthe residue remaining was purified via 3-successive silica gel columnsusing hexanes-ethyl acetate gradient to give compound 114 (121 mg, 59%yield). ¹H NMR (CDCl₃): δH 7.81 (m, 2H), 7.63 (m, 2H), 7.42 (m, 2H),7.33 (m, 2H), 4.78 (s, 2H), 4.43 (m, 2H), 4.37 (t, J=7.65 Hz, 2H), 4.25(m, 2H), 4.18 (m, 2H), 3.67-3.55 (m, 1OH), 3.39 (m, 4H), 2.84 (s, 4H),1.88 (m, 4H), 1.73 (m, 4H), and 1.32 (s, 6H) ppm. The synthesis isillustrated in FIG. 53.

Example 45 Synthesis of Compound 117

A 500 mL round bottom flask equipped with a magnetic stir bar wascharged with compound 68 (7.3 g, 13.8 mmol, pre-dried in a desiccatorovernight), anhydrous dichloromethane (70 mL), cyclohexene (1.54 mL,15.2 mmol) and 3-Å molecular sieves (16.6 g) and the resultingsuspension was stirred for 20 min at 0° C. under a nitrogen atmosphere.Next, SO₂Cl₂ (1 M solution in dichloromethane, 32.7 mL, 2.36 equiv) wasadded and the resulting mixture was stirred at 0° C. for 1 h. Reactionprogress was monitored via TLC for disappearance of the startingmaterial (100% ethyl acetate). Once the SO₂Cl₂ activation was complete,a mixture of (MeO)₃BnSH (7.4 g, 34.5 mmol, 2.5 eqiv) and NaH (1.32 g,33.12 mmol, 60% in mineral oil) in DMF (120 mL) was prepared and rapidlyadded in one portion. The reaction was allowed to slowly warm to roomtemperature and stirred for 1 h. The reaction mixture was filtered andconcentrated in vacuo at 40° C. Purification by column chromatography onsilica gel using 0 to 60% ethyl acetate in hexanes gradient afforded thedesired compound 70 (4.2 g, 43.7% yield) as a clear oil. ¹H NMR (CDCl₃):δ_(H) 8.72 (s, 1H), 8.31 (s, 1H), 7.94 (m, 2H), 7.52 (m, 1H), 7.44 (m,2H), 6.41 (m, 1H), 6.03 (s, 2H), 4.67 (s, 2H), 4.50 (m, 1H), 4.10 (m,1H), 3.73 (m, 13H), 2.52 (m, 2H), 0.81 (s, 9H) and 0.002 (d, 6H) ppm.The synthesis is illustrated in FIG. 54.

Example 46 Synthesis of Compound 71

A 200 mL round bottom flask equipped with a magnetic stir bar wascharged with compound 117 (2.0 g, 2.87 mmol) and dichloromethane (38 mL)under an atmosphere of N2 and cooled on an ice-water bath. To thismixture was added dimethyldisulfide (1.3 mL, 14.36 mmol, 5 equiv),followed by addition of DMTSF (620 mg, 3.15 mmol, 1.1 equiv) as asolution in dichloromethane (20 mL). The resulting mixture was allowedto slowly warm to room temperature and stirred for an additional 4 h.The reaction was then quenched by addition of a saturated aqueoussolution of NaHC₃ (100 mL), extracted with dichloromethane (150 mL×2)and ethyl acetate (200 mL) dried over Na₂SO₄ and concentrated in vacuo.Purification by column chromatography on silica gel (eluted with 0 to60% ethyl acetate in hexanes gradient) gave the desired compound 71 (1.0g, 62%) as a white powder. ¹H NMR (CDCl₃): δ_(H) 8.69 (s, 1H), 8.24 (s,1H), 7.94 (m, 1H), 7.51 (m, 1H), 7.42 (m, 2H), 6.41 (m, 1H), 4.82 (m,2H), 4.57 (m, 1H), 4.15 (m, 1H), 3.77 (m, 2H), 2.61 (m, 2H), 2.40 (s,3H), 0.81 (s, 9H) and 0.00 (d, 6H) ppm. The synthesis is illustrated inFIG. 54.

Example 47 Synthesis of Compound 119

Compound 71 (562 mg, 1.25 mmol) was dissolved in anhydrous THF (30 mL)in a round bottom flask equipped with a stir bar and rubber septum underan atmosphere of N₂ and cooled on an ice-water bath. TBAF (1.5 mL of 1 Msoln. in THF, 1.5 equiv) was then added dropwise and stirred at 0° C.for 2 h. The reaction progress was monitored by TLC (100% ethyl acetateR_(f) for compound 119=0.2, R_(f) for compound 71=0.6). Upon reactioncompletion methanol (5 mL) was added, the reaction was concentrated onthe rotary and the residue was purified via column chromatography onsilica gel (eluted with 0 to 60% ethyl acetate:hexanes gradient 15 mins,followed by 60% ethyl acetate in hexanes for 45 mins) to afford thedesired compound 119 (280 mg, 62% yield) as white powder. ¹H NMR(CDCl₃): δ_(H) 8.69 (s, 1H), 8.02 (s, 1H), 7.95 (m, 2H), 7.53 (m, 1H),7.44 (m, 2H), 6.25 (m, 1H), 4.83 (m, 2H), 4.70 (m, 1H), 4.29 (m, 1H),3.93 (m, 1H), 3.74 (m, 1H), 2.99 (m, 1H), 2.43 (s, 3H) and 2.41 (m, 1H)ppm.

Compound 119 was then converted to triphosphate 120 using the standardtriphosphate synthesis method vide infra, except the de-protection wascarried out by treating with 10% NH₄OH for 5 h at room temperature tominimize —SSMe cleavage. Yield 25%; HRMS-ES⁺: calculated forC₁₂H₂₀N₅O₁₂P₃S₂ 582.976, observed m/z 582.975 The synthesis isillustrated in FIG. 54.

Example 48 Synthesis of Compound 123

Compound 121 (2.5 g, 4.94 mmol) was dried in a desiccator overnight anddissolved in anhydrous dichloromethane (25 mL) in a dry round bottomflask equipped with a stirbar and rubber septum under an atmosphere ofN₂. Cyclohexene (0.55 mL, 1.1 equiv) and dry 3-A molecular sieves (6.0g) were added to the reaction mixture and the resulting suspension wasstirred for 20 min at room temperature. The reaction flask was thenplaced on an ice-salt-water bath to bring the temperature to sub-zeroand SO₂Cl₂ (7.4 mL, 1 M solution in dichloromethane) was added slowlywith a syringe. The resulting mixture was stirred at 0° C. for 1 hfollowed by addition of 0.5 equivalents of SO₂Cl₂ to bring the reactionto completion. Reaction progress was monitored via TLC by thedisappearance of the starting material. Next, a suspension of (MeO)₃BnSH(2.65 g, 12.35 mmol, 2.5 eqiv) and NaH (0.472 g, 11.85 mmol, 60% inmineral oil) in DMF (40 mL) was prepared in a separate flask. Thereaction mixture was combined and slowly warmed to room temperature andstirred for 1 h. The reaction mixture was then filtered through a glasssintered funnel to remove MS, the filtrate was quenched by addition of50 mM aqueous NaH₂PO₄ solution (50 mL) and extracted withdichloromethane. The combined organics were dried over Na₂SO₄ andconcentrated in vacuo. Purification by column chromatography on silicagel using hexanes:ethyl acetate gradient gave the desired compound 123(1.4 g, 42.2% yield). ¹H NMR (CDCl₃): δ_(H) 8.29 (m, 1H), 7.77 (m, 2H),7.48 (m, 1H), 7.38 (m, 2H), 6.15 (m, 1H), 5.99 (m, 2H), 4.55 (m, 2H),4.32 (m, 1H), 4.00 (m, 1H), 3.80 (m, 1H), 3.75 (m, 1H), 3.69 (m, 9H),2.52 (m, 1H), 1.97 (m, 1H), 0.80 (m, 9H) and 0.01 (in, 6H) ppm. Thesynthesis is illustrated in FIG. 55.

Example 49 Synthesis of Compound 124

Compound 123 (1.4 g, 2.08 mmol) was dissolved in anhydrousdichloromethane (42 mL) in a 200 mL round bottom flask equipped withstirbar and a rubber septum under an atmosphere of N2 and cooled to at0° C. To this mixture was added dimethyldisulfide (0.93 mL, 10.4 mmol, 5equiv), followed by addition of DMTSF (450 mg, 2.28 mmol, 1.1 equiv).The resulting mixture was stirred at 0° C. for 2 h. The reaction wasquenched by addition 50 mM NaHCO₃ (100 mL), extracted withdichloromethane (100 mL×2) and dried over Na2SO4 and concentrated invacuo. The product was purified by column chromatography on silica gel(eluted with 0 to 30% ethyl acetate in dichloromethane gradient toafford the desired compound 124 (0.93 g, 83.1%) as a white powder. ¹HNMR (CDCl₃): δ_(H) 8.48 (m, 1H), 7.93 (m, 2H), 7.56 (m, 1H), 7.47 (m,1H), 7.37 (m, 2H) 6.00 (m, 1H), 4.73 (m, 2H), 4.34 (m, 1H), 4.07 (m,1H), 3.84 (m, 1H), 3.73 (m, 1H), 2.44 (m, 1H), 2.33 (m, 3H), 2.25 (m,1H), 0.76 (m, 9H) and 0.01 (m, 6H) ppm. The synthesis is illustrated inFIG. 55.

Example 50 Synthesis of Compound 125

Compound 124 (930 mg, 1.73 mmol) was dissolved in anhydrous THF (52 mL)in a 100 mL round bottom flask equipped with a stirbar and rubber septumunder an atmosphere of N2 and cooled to 0° C. on an ice-water bath. TBAF(3.5 mL of 1 M soln. in THF, 1.5 equiv) was then added drop-wise andstirred at 0° C. for 4 h. Upon reaction completion methanol (5 mL) wasadded to quench the reaction, the volatiles were removed under reducedpressure, and the residue was purified via column chromatography onsilica gel (0 to 75% ethyl acetate in hexanes gradient) to afford thedesired compound 125 (425 mg, 58% yield) as white powder. ¹H-NMR(CDCl₃): δ_(H) 8.24 (m, 1H), 7.81 (m, 1H), 7.51-7.42 (m, 2H), 7.41 (m,2H), 6.09 (m, 1H), 4.80 (m, 2H), 4.50 (m, 1H), 4.17 (m, 1H), 3.94 (m,1H), 3.80 (m, 1H), 2.58 (m, 1H), 2.40 (m, 3H) and 2.41 (m, 1H) ppm. Thesynthesis is illustrated in FIG. 55.

Example 51 Synthesis of Compound 126

Compound 125 was then converted to triphosphate 126 using the standardtriphosphate synthesis procedure vide infra; the final de-protectionstep was carried out by treating with 10% NH₄OH for 2 h at roomtemperature to minimize —SSMe cleavage. 30% yield, HR MS-ES⁺: calculatedfor C₁₁H₂₀N₃O₁₃P₃S₂, 558.965; observed m/z 558.964. The synthesis isillustrated in FIG. 55.

Example 52 Synthesis of Compound 130

A 100 mL round bottom flask equipped with a magnetic stir bar wascharged with 127 (2.0 g, 2.8 mmol) and dried in a desiccator over P₂O₅under high vacuum for 12 h. Dichloromethane (40 mL) was added under N2and the resulting solution cooled on a salt-ice bath for 15 minutes.Cyclohexene (0.34 mL, 3.4 mmol) was added, followed by dropwise additionof SO₂Cl₂ (3.4 mL, 1 M soln. in dichloromethane, 3.4 mmol). Theresulting mixture was stirred for 30 minutes, and the reaction progresswas monitored by TLC (ethyl acetate:hexanes/1:1, 127 R_(f)=0.5, 128R_(f)=0.15 for —CH₂Cl decomposed product). Additional SO₂Cl₂ (3.1 mL, 1M soln. in dichloromethane, 3.1 mmol) was added drop-wise and thereaction mixture was stirred for another 40 minutes to ensure completeconversion to compound 128. This mixture was then concentrated underhigh vacuum at 0° C.

Anhydrous dichloromethane (40 mL) was then added to the residue under N2and the mixture was stirred at 0° C. until all solids dissolved. Asolution of potassium p-toluenethiosulfonate (0.96 g, 425 mmol) in DMF(8 mL) was added slowly and the resulting reaction mixture was stirredat 0° C. for 1 h. The mixture was first concentrated under reducedpressure at 0° C., and then at room temperature. The residue waspurified by flash column chromatography on silica gel column using 0 to100% ethyl acetate in hexanes gradient to give compound 130 as a creamsolid (1.1 g, 51%; TLC R_(f) 0.35, ethyl acetate:hexanes 2:1). MS (ES)m/z: 733 [M+1′]. ¹H NMR (CDCl₃, 400 MHz): δ_(H) 8.02 (br.s, 1H), 7.94(s, 1H), 7.88 (d, J=8.3 Hz, 2H), 7.45 (m, 4H), 7.38 (m, 6H), 7.27 (m,2H), 6.01 (t, J=6.6 Hz 1H), 5.46 & 5.38 (AB, J_(AB)=12.1 Hz, 2H), 4.97(m, 1H), 3.86 (m, 1H), 3.74 (dd, J=12.5, 2.8 Hz, 1H), 3.55 (dd, J=12.5,2.9 Hz, 1H), 2.87 (m, 1H), 2.65 (m, 1H), 2.43 (s, 3H), 2.17 (m, 1H),1.26 (d, J=6.8 Hz, 3H), 1.25 (d, J=6.9 Hz, 3H) ppm. The synthesis isillustrated in FIG. 56.

Example 53 Synthesis of Compound 131

To a solution of 130 (1.1 g 1.5 mmol) in dichloromethane (anhydrous, 40mL) cooled in on an ice-water bath was added dimethyldisulfide (0.66 mL,7.5 mmol) under N2. The resulting mixture was stirred for 15 min andNaSMe (0.23 g, 3.3 mmol) was added in one portion. The resultingreaction mixture was stirred at 0° C. for 4 h (the reaction progress wasmonitored by TLC (ethyl acetate:hexanes/2:1, 130 R_(f)=0.35, 131R_(f)=0.45). The mixture was filtered through Celite-S and concentratedunder reduced pressure. The residue was purified on silica gel column,eluted with ethyl acetate in hexanes (0-100%)) to afford compound 131 asa white solid (0.68 g, 75%; TLC R_(f): 0.45, Ethyl acetate/hexanes/2:1).MS (ES) m/z: 625 [M+1⁺]. ¹H NMR (CDCl₃): δ_(H) 8.02 (s, 1H), 8.00 (br.s, 1H), 7.45 (m, 4H), 7.39 (m, 4H), 7.28 (m, 2H), 6.24 (t, J=6.2 Hz,1H), 5.05 (m, 1H), 4.99 & 4.94 (AB, J_(A)B=11.4 Hz, 2H), 4.27 (m, 1H),3.99 (dd, J=12.5, 2.3 Hz, 1H), 3.86 (dd, J=12.5, 2.3 Hz, 1H), 3.12 (m,1H), 2.74 (m, 1H), 2.52 (s, 3H), 2.50 (m, 1H), 1.30 (d, J6.6 Hz, 3H) and1.29 (m, 3H) ppm. The synthesis is illustrated in FIG. 56.

Example 54 Synthesis of Compound 132

Compound 131 was then converted to triphosphate 132 via standardtriphosphate synthesis method described in standard method section. 25%yield; HRMS-ES⁺: calculated for C₂H₂₀N₅O₁₃P₃S₂, 598.971, observed m/z598.970. The synthesis is illustrated in FIG. 56.

Example 55 Synthesis of Compound 134

Compound 133 (4.47 g, 10.7 mmol) and(2,4,6-trimethoxyphenyl)methanethiol (TMPM-SH) were dried under highvacuum for 2 h and then placed in a desiccator with P₂O₅ for 12 h.Compound 133 was dissolved in anhydrous CH₂Cl₂ (50.0 mL) and cyclohexene(10 mL, 96.6 mmol) was added. The resulting mixture was stirred for 15minutes at −10° C. under a nitrogen atmosphere. Next a freshly preparedsolution of 1 M SO₂Cl₂ in CH₂Cl₂ (25 mL, 26.75 mmol) was added drop-wisevia addition funnel, and the resulting mixture stirred for 1 hour at−10° C. The volatiles were removed in vacuo while keeping the bathtemperature at 10° C. The residue was then dissolved in anhydrous DMF(52 mL) and kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (4 g, 18.7mmol) was dissolved in anhydrous DMF (48 mL) under a nitrogen atmosphereand cooled to 0° C. NaH (1.1 g, 26.8 mmol, 60% in mineral oil) was thenadded and the resulting grey slurry was stirred for 15 minutes at 0° C.It was added to the former solution in one portion and the reaction wasstirred at room temperature for 1 h. The reaction mixture was thenpartitioned in a reparatory funnel (150:300 mL/brine:ethyl acetate). Theorganic layer was then washed with brine (2×150 mL). The aqueous layerwas back-extracted (4×50 mL ethyl acetate). The combined organic layerwas dried over anhydrous sodium sulfate. The solvent was removed andproduct was purified by flash chromatography on silica gel column(column: 120 g RediSepRfGold-ISCO, gradient 0-100% ethyl acetate inhexanes). The target compound 134 was obtained as white solid in 22%yield (1.35 g). ¹H NMR (CDCl₃): δ_(H) 8.17 (s, 1H), 7.39 (d, 1H), 6.30(m, 1H), 6.12 (s, 2H), 4.71 (dd, 2H), 4.43 (m, 1H), 4.04 (m, 1H), 3.87(m, 1H), 3.83 (m, 9H), 3.74 (dd, 1H), 2.74 (ddd, 1H), 2.34 (ddd, 1H),1.93 (m, 2H) 1.53 (s, 3H), 0.93 (m, 9H), 0.11 (m, 6H) ppm. LCMS (ESI)[M−H⁺] observed 581, R_(f)=0.59 (4:6/hexanes-ethyl acetate). Andcompound 135 was also isolated as aside product in 22.5% yield (1.13 g).¹H NMR (CDCl₃): δ_(H) 8.55 (s, 1H), 7.41 (m, 1H), 6.12 (M, 3H), 4.76(dd, 2H), 4.47 (m, 1H), 4.01 (m, 1H), 3.90 (m, 1H), 3.82 (m, 9H), 3.75(m, 1H), 2.29 (m, 2H), 2.04 (s, 3H) and 1.91 (m, 2H) ppm. LCMS (ESI)[M−H⁺] observed 467. The synthesis is illustrated in FIG. 57.

Example 56 Synthesis of Compound 136

Compound 134 (3.6 g, 6.2 mmol) in a 100 mL round bottom flask was driedunder high vacuum for 2 h and then placed in a vacuum desiccator withP₂O₅ for 12 h. Anhydrous CH₂Cl₂ (96 mL) and dimethyldisulfide (2.8 mL,30.9 mmol) were added, and the reaction cooled to 0° C.Dimethyl(methylthio)sulfonium tetrafluoroborate (DMTSF, 1.34 g, 6.82mmol) was then added and the reaction stirred for 1 h at 0° C. Thereaction mixture was next transferred to a 250 mL separatory funnel andneutralized with 90 mL of 0.1 M aqueous solution of NaHCO₃, andextracted with ethyl acetate (2×200 mL). Combined organic layer wasdried over anhydrous sodium sulfate and concentrated on the rotary. Theresidue was purified by flash chromatography on a silica gel columnusing 30-50% ethyl acetate in hexanes gradient. The target compound 136was obtained as white solid (2.1 g, 77% yield). ¹H NMR (CDCl₃): δ_(H)7.99 (s, 1H), 7.47 (d, 1H), 6.29 (dd, 1H), 4.87 (dd, 2H), 4.49 (m, 1H),4.13 (m, 1H), 3.88 (m, 2H), 3.5 (m, 1H), 2.47 (s, 3H), 2.45 (dd, 1H),2.04 (dd, 1H) and 1.54 (s, 2H), 0.93 (m, 9H) and 0.13 (m, 6H) ppm. LCMS(ESI) [M−H⁺] observed 447.0. The synthesis is illustrated in FIG. 57.

Example 57 Synthesis of Compound 137

Compound 136 (2.16 g, 4.8 mmol) in a 100 mL round bottom flask driedunder high vacuum for 2 h, was dissolved in anhydrous THF (40 mL)followed by addition of acetic acid (1.2 mL) and TBAF in THF (6.7 mL of1 M solution, 6.72 mmol). The reaction mixture was stirred for 1 hour at0° C. and then for 2 additional hours at room temperature. The volatileswere removed in vacuo and the residue purified via flash chromatographyon 40 g RediSepRf gold column using 0-8% Methanol in dichloromethanegradient. The target compound 137 was obtained as white solid (1.45 g,90% yield). ¹H NMR (CDCl₃): δ_(H) 8.12 (s, 1H), 7.36 (d, 1H), 6.11 (t,1H), 4.87 (dd, 2H), 4.57 (m, 1H), 4.14 (q, 1H), 3.94 (dd, 1H), 3.83 (m,1H), 2.50 (s, 3H), 2.4(m, 2H), 1.93 (s, 3H) ppm; LCMS (ESI) [M−H⁺]observed 333. The synthesis is illustrated in FIG. 57.

Example 58 Synthesis of Compound 138

The product 138 was obtained after phosphorylation of compound 137 usingthe standard triphosphate synthesis method vide infra. 40% yield, HRLC-MS: calculated for C₁₂H₂₁N₂O₁₄P₃S₂, 573.965; observed m/z 573.964.The synthesis is illustrated in FIG. 57.

Example 59 Synthesis of Compound 141

A 100 mL round bottom flask equipped with a magnetic stir bar wascharged with compound 139 (2.23 g, 3.55 mmol), CH₂Cl₂ (20 mL), 3-Åmolecular sieves (3.5 g) and cyclohexene (0.60 mL). The resultingmixture was stirred for 20 minutes at room temperature under a nitrogenatmosphere. The reaction was cooled to 0° C. and SO₂Cl₂ (5.4 mL, 1 M inCH₂Cl₂, 1.5 equiv) were added slowly via a syringe. The reaction wasstirred for 1.5 h at 0° C. and an additional 1.8 mL of SO₂Cl₂ (1 M soln.in dichloromethane) was added and stirring continued for 40 minutes at0° C. to ensure complete conversion to compound 140. The volatiles wereremoved under reduced pressure while keeping the bath temperature closeto 10° C. The resulting solid was re-suspended in 20 mL of anhydrous DMFand kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (1.98 g, 9.25mmol) was dissolved in anhydrous DMF (15 mL) and treated with NaH (247mg, 60% in mineral oil, 6.17 mM) producing a dark grey slurry. Next,compound 140 solution was added in one portion and the reaction wasstirred at room temperature for 1 h. The reaction mixture was thenpartitioned between distilled water (150 mL) and ethyl acetate (150 mL).The organic layer was further washed with distilled water (2×150 mL) anddried over Na₂SO₄. The volatiles were removed under reduced pressure andthe residue was purified by flash column chromatography on silica gelcolumn using 80 to 100% ethyl acetate in hexanes gradient. The targetcompound 141 was obtained as white solid (798 mg, 28%). ¹H NMR (CDCl₃):δ_(H) 8.33 (s, 1H), 7.57 (m, 1H), 6.53 (m, 2H), 6.00 (s, 2H), 4.62 (m,2H), 4.44 (m, 1H), 4.32 (m, 2H), 3.97 (m, 1H), 3.80-3.60 (m, 11H), 3.10(m, 6H), 2.36 (m, 1H), 2.24 (m, 1H), 0.80 (m, 9H) and 0.01 (m, 6H) ppm.Further confirmed by LC-MS: observed m/z 795.25 for (M−H). The synthesisis illustrated in FIG. 58.

Example 60 Synthesis of Compound 142

A 100 mL round bottomed flask equipped with a magnetic stir bar wascharged with compound 141 (0.779 gm, 0.98 mmol, vacuum dried over P₂O₅for 12 h) and dry THF (20.0 mL), and cooled to 0° C. under a nitrogenatmosphere. TBAF (1.17 mL, 1M solution in THF, 1.17 mmol) was addedslowly via a syringe and the reaction mixture was stirred for 1.5 h at0° C. Next, an additional TBAF (1 mL, 1M solution in THF, 1 mmol) wasadded and reacted for 3 h at 0° C. The reaction mixture was thentransferred to a separatory funnel and quenched by addition of methanol(5 mL), distilled water (100 mL) was added and the reaction extractedwith ethyl acetate (2×100 mL). The organics were dried over Na₂SO₄ andconcentrated in vacuo. Column chromatography of the residue on silicagel using 80-100% ethyl acetate in hexanes gradient afforded compound142 as a white powder (525 mg, 79%). ¹H NMR (Methanol-d4): δ_(H) 8.33(s, 1H), 7.19 (m, 1H), 6.06 (m, 2H), 6.03 (m, 1H), 4.72 (m, 2H), 4.64(m, 1H), 4.57 (m, 1H), 4.35 (m, 2H), 4.17 (m, 1H), 3.75 (m, 9H), 3.16(m, 6H), 2.80 (m, 1H) and 2.28 (m, 1H) ppm; LC-MS: M−H observed m/z680.0. The synthesis is illustrated in FIG. 58.

Example 61 Synthesis of Compound 143

Compound 143 was synthesized from compound 142 via standard triphosphatesynthesis procedure described in the standard methods section. Yield65%, LRMS-ES⁻: calculated for C₂₅H₃₃N₅O₁₅P₃S—, 768.09; observed m/z768.54 (M−H). The synthesis is illustrated in FIG. 58.

Example 62 Synthesis of Compound 144

A 50 mL conical tube was charged with compound 143 (3.80 mL of 5.25 mMsoln. in HPLC grade water, 20 μmols) and pH 4.65 acetate buffer (4.75mL), and quickly combined with 9.0 mL of freshly prepared DMTSF (80 mM)solution in pH 4.65 acetate buffer. The resulting mixture was shaken atroom temperature for 2 h and quenched by addition of saturated aqueoussolution of NaHCO₃ (2 mL). The product was immediately purified onpreparative HPLC (column: 30×250 mm C₁₈ Sunfire, method: 0 to 2.0 min100% A, followed by 50% B over 70 min, flow: 25 mL/min, A=50 mM TEAB,B=acetonitrile). The appropriate fractions were lyophilized and combinedafter dissolving in HPLC grade water to furnish 23.4 umols of compound144 (73% yield). LRMS-ES⁻: calculated for C₆H23N₅O12P₃S2-, 634.00, m/zobserved 634.42 for (M−H). The synthesis is illustrated in FIG. 58.

Compound 144 was converted to dye labeled product (76) according toprocedure described in standard methods section (FIG. 59). Compound 146was obtained in 75% yield in two steps, LRMS-ES⁺: calculated forC₃₄H₅₉N₇O₁₉P₃S₄,1090.20, m/z observed 1090.24 for (M+H). Compound 76 wasobtained in 50-70% yield from 146, HRMS-ES⁻: calculated forC₆₇H₈₆N₉O₂₃P₃S₄,1605.393; observed m/z 1605.380 for (M−H).

Example 63 Synthesis of Compound 150

A 250 mL round bottom flask was charged with compound 148 (3.0 g, 4.58mmol), 25 mL dry CH₂Cl₂, 3-Å molecular sieves (5.0 g) and cyclohexene(0.55 mL, 5.4 mmol). The resulting mixture was stirred for 10 minutes atroom temperature under a nitrogen atmosphere. The reaction flask wasthen placed on an ice-bath, SO₂Cl₂ (6.8 mL, 1M in CH₂Cl₂, 1.5 eq) wasadded slowly via a syringe, and the reaction stirred for 1 h at 0° C.Next, an extra 0.5 eq of SO₂Cl2 was added to ensure complete conversionto compound 149. The volatiles were removed under vacuum while keepingthe temperature close to 10° C. The resulting solid was re-suspended in20 mL of dry DMF and kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (2.45 g, 11.44mmol) was dissolved in dry DMF (30 mL) under nitrogen atmosphere, andtreated with NaH (274.5 mg, 60% in silicon oil) producing a grey slurry.To this, compound 149 was added at once and the reaction stirred at roomtemperature for 3 h under nitrogen atmosphere. The reaction mixture wasthen filtered through Celite®-S washed with ethyl acetate (100 mL). Theethyl acetate solution was washed with distilled water (2×100 mL), theorganic extract was dried over Na₂SO₄. concentrated in vacuo andpurified via flash column chromatography on silica gel column using 20to 50% ethyl acetate in hexanes gradient. The target compound 150 wasobtained as white solid (1.2 g, 32% yield, R_(f) 0.4, hexanes:ethylacetate/3:2). ¹H NMR (CDCl₃): δ_(H) 8.13 (m, 3H), 7.43 (m, 1H), 7.32 (m,2H), 6.12 (m, 1H), 6.00 (s, 2H), 4.62 (m, 2H), 4.31 (m, 3H), 4.00 (m,1H), 3.82-3.60 (m, 13H), 2.39 (m, 1H), 1.84 (m, 1H), 0.78 (m, 9H), and0.01 (m, 6H) ppm. The synthesis is illustrated in FIG. 60.

Example 64 Synthesis of Compound 151

Compound 150 (1.2 g 1.46 mmol) was dried under high vacuum with P₂05 ina desiccator overnight and dissolved in 30 mL of anhydrous CH₂Cl₂ in a100 mL flask equipped with a magnetic stir bar. To this was addeddimethyldisulfide (0.657 mL, 7.3 mmol), and the reaction flask wasplaced on an ice-bath. Dimethyl(methylthio)sulfonium tetrafluoroborate(DMTSF, 316 mg, 1.1 eq) was added and stirred for 1.5 hr at 0° C. Thereaction mixture was transferred to a 250 mL separatory funnel andneutralized with 50 mL of 0.1 M aq. solution of NaHC₃, and extractedwith CH₂Cl₂ (2×50 mL). The organic layer was dried over Na₂SO₄ andconcentrated by rotary evaporation. The crude product was purified on asilica gel column using gradient 80-50% ethyl acetate in hexanesgradient to result in 0.82 g of compound 151 (82% yield, R_(F)=0.5,hexanes:ethyl acetate/3:2). ¹H NMR (CDCl₃): δ_(H) 8.15 (m, 3H), 7.42 (m,1H), 7.35 (m, 2H), 6.11 (m, 1H), 4.80-4.65 (m, 2H), 4.34 (m, 1H), 4.28(m, 2H), 4.10 (m, 1H), 3.83-3.67 (m, 2H), 2.49 (m, 1H), 2.34 (s, 3H),1.90 (m, 1H), 0.78 (m, 9H), and 0.10 (m, 6H) ppm.

The synthesis is illustrated in FIG. 60.

Example 65 Synthesis of Compound 152

A 100 mL round bottomed flask equipped with a magnetic stir bar wascharged with compound 151 (0.309 g, 0.45 mmol), and 10.0 mL dry THF(10.0 mL) and placed on an ice-bath under a nitrogen atmosphere. TBAF(0.72 mL, 1 M soln. in THF, 0.72 mmol) was added slowly via syringe. Thereaction mixture was stirred for 3 h at 0° C. The reaction mixture wasthen transferred to a separatory funnel and quenched with 0.5 M aqueoussoln. of NaHCO₃ (50 mL). The resulting mixture was then extracted withethyl acetate (2×100 mL) and dried over Na₂SO₄. The product 152 wasobtained as a white powder after silica gel column chromatography in 76%yield (196 mg, R_(f)=0.3, hexanes:ethyl acetate/1:1) on silica gelcolumn using gradient 7:3 to 2:3 hexanes:ethyl acetate. ¹H NMR (CDCl₃):δ_(H) 8.40 (s, 1H), 8.25 (m, 2H), 7.60 (m, 1H), 7.52 (m, 2H), 6.21 (m,1H), 4.90-80 (m, 2H), 4.65 (m, 1H), 4.40 (m, 2H), 4.25 (m, 1H),4.05-3.85 (m, 2H), 2.62 (m, 1H), 2.50 (s, 3H) and 2.31 (m, 1H) ppm. Thesynthesis is illustrated in FIG. 60.

Example 66 Synthesis of Compound 153

Compound 153 was obtained after phosphorylation of compound 152 in 30%yield using the standard triphosphate synthesis method vide infra(LC-MS: calculated for C₁₄H23N₄O₃P₃S₂. 610.98; observed m/z 611.11(M−H). It was further converted to dye labeled product (72) according toprocedure described in standard method section (FIG. 61). Compound 155was obtained in 49% yield in two steps, and compound 72 in 60-85% yield,HRMS-ES⁻: calculated C₅₃H₆₈N₈SO₃₀P₃S₆ ⁻, 1581.156 (M−H); found m/z1582.160.

Example 67 Synthesis of compounds 159 & 160

A 100 mL round bottom flask equipped with a magnetic stir bar wascharged with compound 157 (2.04 g, 2.39 mmol) and was dried on highvacuum over 12 h. After flushing the reaction vessel with argon, 13 mLanhydrous CH₂Cl₂ and cyclohexanesene (0.30 mL, 2.86 mmol) were addedsequentially. The reaction flask was then placed on an ice-water-saltbath and stirred for 10 min to bring the mixture below 0° C. S₂Cl₂ (4.0mL, 1M in CH₂Cl₂, 4.0 mmol) was added drop-wise via a syringe over 2min, and the reaction mixture stirred for 1 h at 0° C. An additional 0.8equiv. of SO₂Cl₂ (2.0 mL, 2.0 mmol) was added drop-wise over 1 min andthe reaction was stirred for an additional %2 h at 0° C. Next, thevolatiles were removed in vacuo while keeping the bath temperature at10° C. The resulting solid was re-suspended in 15 mL of dry DMF and keptunder an argon atmosphere.

In a separate 100 mL flask, (2,4,6-trimethoxyphenyl)methanethiol(TMPM-SH, 1.27 g, 6.0 mmol, vacuum dried overnight) was dissolved in dryDMF (16 mL) under argon atmosphere and treated with NaH (195 mg, 60% inoil, 4.88 mmol) producing a grey slurry TMPMT-SNa salt. The mixture wasstirred until gas formation subsided (Ca. 10 min). To this, TMPMT-SNasalt was added at once and the mixture was stirred at room temperatureunder argon atmosphere until TLC (micro-workup: dichloromethane/water;solvent:hexanes:ethyl acetate/1:1) confirmed complete conversion (1 h).The reaction mixture was then filtered through Celite®-S (10 g) in afiltration funnel eluting the product with dichloromethane (100 mL). Thedichloromethane solution was then washed with water (3×100 mL). Theaqueous layer was extracted with 3×100 mL dichloromethane. Combineddichloromethane extract was dried over Na₂SO₄ and concentrated by rotaryevaporation. It was then purified by flash chromatography (column: 100g, gradient: 25%-50% hexanes:ethyl acetate 5 CV, then 50% EE 10 CV). Thetarget compound 160 was obtained as a white foam (1.22 g, 51% yield). ¹HNMR (DMSO-d): δ_(H) 10.63 (s, 1H), 10.15 (s, 1H), 7.95 (s, 1H), 7.3-7.5(m, 8H), 7.20-7.3 (m, 2H), 6.40 (m, 1H), 6.15 (m, 1H), 4.69 (m, 2H),4.50 (dd, 1H), 4.30 (m, 2H), 3.95 (m, 1H), 3.81 (m, 11H), 3.3 (m, 4H),2.7 (m, 1H), 1.05 (m, 8H), 0.8 (m, 9H) and 0.11 (m, 6H) ppm. LCMS:1019.371 Da. The synthesis is illustrated in FIG. 62.

Additionally, the TBDMS-deprotected product 159 was obtained as a sideproduct in 25% yield (0.48 g). R_(f)=0.2/hexanes:ethyl acetate/1:1. ¹HNMR (DMSO-d₆): δ_(H) 10.63 (s, 1H), 10.15 (s, 1H), 7.95 (s, 1H), 7.3-7.5(m, 8H), 7.20-7.3 (m, 2H), 6.40 (m, 1H), 6.15 (m, 1H), 4.69 (m, 2H),4.50 (dd, 1H), 4.30 (m, 2H), 3.95 (m, 1H), 3.81 (m, 11H), 3.5 (m, 1H),3.3 (m, 4H), 2.7 (m, 1H), and 1.04 (m, 8H) ppm. LCMS: 905.286 Da.

Example 68 Synthesis of Compound 161

A 100 mL round bottom flask equipped with a magnetic stir bar and rubberseptum was charged with compound 160 (0.36 g, 0.35 mmol) and dried for12 h on high vacuum. After flushing with argon, 7 mL dry dichloromethaneand dimethyldisulfide (0.16 mL, 1.76 mmol) were added. The reactionflask was placed on an ice-bath and stirred for 10 min to bring themixture to 0° C. Dimethyl(methylthio)sulfonium tetrafluoroborate (DMTSF,80 mg, 0.4 mmol) was then added and the reaction was stirred for at 0°C. until TLC (micro-workup:dichloromethane/water; solvent: Hexanes:Ethylacetate/1:1). The reaction mixture was transferred to a 250 mLseparatory funnel, neutralized with 50 mL of 0.1 M aq. solution ofNaHCO₃ and extracted with CH₂Cl₂ (3×50 mL). The organic layer was driedover Na₂SO₄ and concentrated by rotary evaporation. The crude productwas purified on a silica gel column (column: 25 g, gradient: 10%-50%hexanes:ethyl acetate 3 CV, then 50% ethyl acetate 5 CV). The targetcompound 161 was obtained as yellow foam (0.23 g, 74% yield). Thesynthesis is illustrated in FIG. 62.

Example 69 Synthesis of Compound 162

A 100 mL round bottomed flask equipped with a magnetic stir bar wascharged with compound 161 (0.18 g, 0.20 mmol), dissolved in 7.0 mL dryTHF and placed on an ice-bath under an argon atmosphere. The mixture wasstirred for 10 min to bring it to 0° C. and 0.28 mL Acetic acid wereadded. TBAF (1 M in THF, 0.47 mL, 0.47 mmol) was added dropwise viasyringe over 1 min. The reaction mixture was stirred for 0.5 h at 0° C.and then 1 h at rt. TLC (hexanes:ethyl acetate/1:1) still showedstarting material. Additional TBAF (1 M in THF, 0.47 mL, 0.47 mmol) wasadded dropwise via syringe over 1 min and the reaction mixture wasstirred for 1 h at room temperature. Next, the mixture was quenched with2 mL methanol and stirred for 10 min at rt. The solvent was removed byrotary evaporation, and the crude product was purified by silica gelcolumn chromatography (column: 10 g, hexanes:ethyl acetate/1:1 to 100%over 2 CV, then 100% Ethyl acetate over 20 CV to yield compound 162 as awhite foam (96 mg, 62%). The synthesis is illustrated in FIG. 62.

Example 70 Synthesis of Compound 163

Compound 163 was obtained after phosphorylation of compound 162 usingthe standard triphosphate synthesis method vide infra; except in thede-protection step AMA or methanolic ammonia were used instead ofammonium hydroxide. It was further converted to the dye labeled product78 according to the standard procedure below. Compound 78 was obtainedin 97% yield from compound 165. HRMS-ES⁻ calculated C₆₇H₉₆N₉O₂₇P₃S₆(M−H) 1743.395, found 1743.390. The synthesis is illustrated in FIG. 62.

Example 71 Synthesis of Compound 169

A 100 mL round bottom flask was charged with compound 167 (3.120 g, 5.66mmol), 30.0 mL dry CH₂Cl₂, 3-Å molecular sieves (5.0 g) andcyclohexanesene (0.70 mL, 6.9 mmol). The resulting mixture was stirredfor 10 minutes at room temperature under a nitrogen atmosphere. Thereaction flask was then placed on an ice-bath. To this, SO₂Cl₂ (8.5 mL,1M in CH₂Cl₂, 1.5 equiv) was added slowly via a syringe, and stirred for1 hour at 0° C. Next, an additional 4.0 mL of 1 M S2Cl₂ was added andstirred for 40 minutes to ensure complete conversion to compound 168.The volatiles were removed under vacuum while keeping the temperatureclose to 10° C. The resulting solid was re-suspended in 20 mL of dry DMFand kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (3.028 g,14.15 mmol) was dissolved in dry DMF (40 mL) under nitrogen atmosphere,and treated with NaH (566 mg, 60% in oil, 14.15 mM) producing a greyslurry. To this, compound 168 solution was added at once and stirred atroom temperature for 2.5 h under nitrogen atmosphere. The reactionmixture was then filtered through Celite®-S (20 g) with ethyl acetate(200 mL). The ethyl acetate solution was then washed with distilledwater (3×200 mL) and dried over Na₂SO₄. concentrated by rotaryevaporation, and purified by flash chromatography on 120 gRediSepRfGold, gradient: hexanes:ethyl acetate (7:3 to 3:7). The targetcompound (169) was obtained as white solid (1.43 g, 35.5% yield, R_(f):0.5, hexanes:ethyl acetate/1:1). ¹H NMR (CDCl₃): δ_(H) 7.98 (m, 1H),6.09 (m, 1H), 6.00 (m, 2H), 4.67-4.51 (m, 2H), 4.30 (m, 1H), 4.22 (m,2H), 4.00 (m, 1H), 3.80-3-60 (m, 11H), 2.31 (m, 1H), 1.83 (m, 1H), 0.80(m, 9H) and 0.01 (in, 6H) ppm. The synthesis is illustrated in FIG. 64.

Example 72 Synthesis of Compound 170

Compound 169 (1.43 g 1.99 mmol) was dried under high vacuum over P₂O₅for 12 h and dissolved in of anhydrous CH₂Cl₂ (25 mL) in a flaskequipped with a magnetic stir bar and a nitrogen gas source. To this wasadded dimethyldisulfide (0.89 mL, 9.88 mmol), and the reaction flask wasstirred on an ice-bath. Dimethyl(methylthio)sulfonium tetrafluoroborate(DMTSF, 430 mg, 2.19 mmol) was then added and stirred for 1.0 h at 0° C.The reaction mixture was transferred to a 500 mL separatory funnel andquenched with 100 mL of 50 mM aq. solution of NaHC₃, and extracted withCH₂Cl₂ (2×150 mL). The organic portion was dried over Na₂SO₄ andconcentrated by rotary evaporation. The crude product was purified on asilica gel column (80 g RediSepRf gold) using hexanes-ethyl acetate (8:2to 3:7) gradient to result in 0.622 gm of compound 170 (54% yield,R_(F)=0.6, hexanes:ethyl acetate/1:1). ¹H NMR (CDCl₃): δ_(H) 7.99 (brs,1H, NH), 7.98 (s, 1H), 6.12 (m, 1H), 4.69 (m, 2H), 4.35 (m, 1H), 4.19(m, 2H), 4.06 (m, 1H), 3.80 (m, 1H), 3.60 (m, 2H), 2.40 (m, 1H), 2.33(s, 3H), 1.88 (m, 1H), 0.78 (m, 9H), and 0.10 (m, 6H) ppm. The synthesisis illustrated in FIG. 64.

Example 73 Synthesis of Compound 171

A 100 mL round bottomed flask equipped with a magnetic stir bar wascharged with compound 170 (0.623 g, 1.06 mmol, vacuum dried over P₂O₅for 12 h) and anhydrous THF (20.0 mL) and placed on an ice-bath under anitrogen atmosphere. TBAF (1.27 mL, 1 M solution in THF, 1.27 mmols) wasadded slowly via syringe. The reaction mixture was stirred for 1.5 h at0° C., and an additional 0.9 mL of 1 M TBAF soln. in THF was added andstirred a total of 4 h at 0° C. The reaction mixture was thentransferred to a separatory funnel and quenched with 0.5 M NaHC₃solution (50 mL). The resulting mixture was extracted with ethyl acetate(2×100 mL) and dried over Na₂SO₄. The product 171 was obtained as awhite powder after silica gel column chromatography in 63% yield (311mg) on a 80 g RediSepRf column using gradient 7:3 to 3:7 ethyl acetatein hexanes. ¹H NMR (methanol-d4): δ_(H) 8.16 (s, 1H), 6.06 (m, 1H), 4.79(m, 2H), 4.69 (m, 1H), 4.40 (m, 1H), 4.14 (m, 2H), 3.99 (m, 1H), 3.63(m, 2H), 2.36 (m, 3H), 2.32 (m, 1H), and 2.08 (m, 1H) ppm, LRMS-ES−: M−Hobserved m/z 468.0 Da. The synthesis is illustrated in FIG. 64.

Example 74 Synthesis of Compound 172

The product 172 was obtained in 58% yield after phosphorylation ofcompound 171 using via standard triphosphate synthesis method. LRMScalculated C₁₄H₂₁N₃O₁₄P₃S₂ ⁻ (M−H), 611.97, found 612.15. Compound 171was further elaborated to the dye labeled product (74) according tostandard procedure described in standard method section vide infra (FIG.65). Compound 174 was obtained in 74% yield in two steps(HRMS-ES-calculated C₃₂H₅₅N₅O₂₁P₃S₄ ⁻ (M−H) 1066.15, found 1066.42.Compound 74 was obtained in 62% yield (HRMS-ES-calculatedC₅₉H₈₀N₇O₂₅P₃S₄ ⁻ (M−H), 1507.330, found 1507.325.

Example 75 Standard Method for Triphosphate Synthesis:

Nucleoside (160 μmol) and proton sponge (1.5 equiv) pre-dried under highvacuum over P₂O₅, were dissolved in trimethylphosphate (0.8 mL) in a 25mL pear-shaped flask under N2-atmosphere and stirred for 20 minutesuntil all solids were completely dissolved. The flask was then placed onan ice-water bath to bring the reaction to (−5 to 0° C.). Then, POCl₃(1.5 eq.) was added in one portion via syringe and the reaction stirredfor 1 h.

A mixture of n-butylammonium-pyrophosphate (0.36 g), n-Bu₃N (0.36 mL)and anhydrous DMF (1.3 mL) was prepared in a 15 mL conical tubeproducing a thick slurry. Once completely dissolved, it was rapidlyadded at once to the vigorously stirring mixture and stirred for 15 minsat room temperature.

The reaction mixture was then poured into 100 mL of 0.1 M TEAB buffer ina 250 mL round bottom flask and stirred for 3 h at room temperature. Itwas then concentrated down to 25 mL in vacuo and treated with 25 mL ofammonium hydroxide (28-30% NH₃ content) for 8 h at room temperature.After removing most of the volatiles under reduced pressure, thereaction crude was resuspended in 0.1M TEAB buffer (30 mL) and purifiedby C18 preparative—HPLC (30×250 mm, C18 Sunfire column, method: 0 to 2min 100% A, followed by 50% B over 70 mins, flow 25 mL/min; A=50 mMTEAB, B=ACN). The target fractions were lyophilized, and combined afterdissolving in HPLC grade water (20 mL). This semi-pure product wasfurther purified by ion exchange HPLC on PL-SAX Prep column (method: 0to 5 min 100% A, then linear gradient up to 70% B over 70 min, whereA=15% acetonitrile in water, B=0.85 M TEAB buffer in 15% acetonitrile).Final purification was carried out by C18 Prep HPLC as described above.The nucleoside triphosphates were obtained in 20-65% yield followinglyophilization.

Example 76

Standard Method for Converting of3′—OCH₂S-(2,4,6-Trimethoxyphenyl)Methane-dNTP to 3′-(OCH₂SSMe)-dNTPUsing DMTSF:

A 50 mL conical tube was charged with3′—OCH₂S-(2,4,6-trimethoxyphenyl)methane-dNTP (3.80 mL of 5.25 mMolarsoln. in HPLC grade water, 20 μmols) and pH=4.65 acetate buffer (5.20mL), and quickly combined with 9.0 mL of DMTSF (80 mMolar soln. inpH=4.65 acetate buffer). The resulting mixture was shaken at roomtemperature for 2 h and the reaction was quenched by 2.0 mL of saturatedNaHCO₃ solution, and immediately purified by prep-HPLC on 30×250 mm C18Sunfire column, method: 0 to 2.0 min 100% A, followed by linear gradientup to 50% B over 70 min, flow: 25 mL/min, A=50 mM TEAB, B=acetonitrile.The target fractions were lyophilized and combined after dissolving inHPLC grade water to result in 50-75% yield of 3′-(OCH₂SSMe)-dNTPdepending on nucleotide. Structrual exampls of3′—OCH₂S-(2,4,6-trimethoxyphenyl)methane-dNTPs are illustrated in FIG.66.

Example 77 Standard Method for Conjugation of NHS Activated Linker:

MeSSdNTP-PA (10 ummol) dissolved in HPLC grade water (2 mL) was dilutedwith freshly prepared 0.5 M aqueous soln. of Na₂HPO₄ (1 mL). In aconical tube, the NHS-activated linker (NHS-A-Fmoc, 114, 35 mg, 2.5 eq.)was dissolved in anhydrous DMF (2.0 mL). It was then added to theMeSSdNTP-PA/Na₂HPO₄ solution at once and stirred for 8 h at roomtemperature.

The reaction was then diluted with 0.1 M TEAB buffer (2.0 mL) andtreated with piperidine (0.6 mL). The mixture was stirred at roomtemperature for 1 h, diluted further with 0.1 M TEAB (10 mL) and quicklypurified by prep HPLC on 30×250 mm C18 Sunfire column, method: 0 to 2.0min 100% A, followed by linear gradient up to 50% B over 70 min, flowrate: 25 mL/min, A=50 mM TEAB, B=acetonitrile. The target fractions werelyophilized and combined after dissolving in HPLC grade water resultingin 45-75% yield of MeSSdNTP-A-NH₂.

Example 78

Standard Method for Labeling with NHS Dye:

MSSdNTP-A-NH2 (4.55 smol) in 2.0 mL of HPLC grade water was diluted withNa₂HPO₄ (0.8 mL of 0.5 Molar aqueous soln.) in a 15 mL conical tube, andcombined with NHS-activated dye (2.5 eq.) in 1.4 mL of anhydrous DMF.The reaction mixture was stirred for 8 h at room temperature, dilutedwith 0.1 M TEAB buffer (40 mL) and purified by prep-HPLC on 30×250 mmC18 Sunfire column, method: 0 to 5 min 100% A, followed by lineargradient up to 50% B over 70 mins, flow rate 25 L/min). The targetfractions were lyophilized and combined after dissolving in HPLC gradewater to result in 50-80% yield of labeled product.

Example 79 Attachment of Cleavable Linkers and Markers to Nucleobases

One of the preferred moieties used to attach cleavable linkers ispropargyl based or allyl based. Other means of attaching cleavablelinkers and dyes are also contemplated. In particular, attachments tothe base moiety that result with little or no residual linker after dyecleavage are particularly preferred. Attachments to the base that resultwith residual linkers after cleavage that do not carry charge are alsopreferred. These features are important to ensure that the nucleotidesare incorporated in the efficient manner by the enzyme into growingstrand of nucleic acid after the cleavage of the label/dye. Oneparticular embodiment contemplated by the present invention comprisesthe use of hydroxymethyl modified base moieties to attach cleavabledyes. Examples of such compounds are shown in FIG. 71. FIG. 72 shows thestructures of hydroxymethyl derivatives after cleavage of the dye andthe 3′-O protective group.

Example 80 Cleavage of Cleavable Linkers and 3′-O Protective Groups

A variety of cleaving agents can be used to cleave the linkers andprotective groups of the present invention. For example, a variety ofthiol carrying compounds can be used as described in (“Thiol-DisulfideInterchange”, Singh, R., and Whitesides, G. M., Sulfur-ContainingFunctional Groups; Supplement S, Patai, S., Eds., J. Wiley and Sons,Ltd., 1993. p 633-658,) [15]. In particular compounds with reduced thiolgroups pKas can be used to achieve fats and efficient cleavage yields,for example dithiobutylamine, DTBA (Lukesh et. al., J. Am. Chem. Soc.,2012, 134 (9), pp 4057-4059 [16]). Examples of thiol bearing compoundsthat can be used to perform cleavage of the current invention are shownin FIG. 73A-I.

Another class of compounds that are suitable for cleaving the dithioterminating groups and linkers of the present invention are phosphines(Harpp et al., J. Am. Chem. Soc. 1968 90 (15) 4181-4182 [12], Burns etal., J. Org. Chem. 1991, 56, 2648-2650 [13], Getz et al., AnalyticalBiochemistry 273, 73-80 (1999) [14]). Examples of phosphines useful tocleave dithio based protective groups and linkers of the presentinvention include: triphenylphosphine, tributylphosphine,tris-hydroxymethyl-phosphine (THMP), tris-hydroxypropyl-phosphine(THPP), tris-carboethoxy-phosphine (TCEP). In certain cases it may bedesired to be able to selectively cleave either the linker or the3′-protective group selectively. This can be achieved by designingprotective group and linker as well as selection of cleavage reagents.For example, a combination of 3′-azidomethyl ether protecting group anddisulfide linker bearing nucleotide can be used for this purpose. Inthis case, selective cleavage of the disulfide bridge can beaccomplished by using thiol based cleaving reagent and removal of3′-azidomethyl ether protecting groups can be achieved by usingphosphine such as TCEP. Example of such procedure is illustrated in FIG.74A-C, FIG. 75A-C, and FIG. 76. FIG. 74A-C shows schemes of chemicalreactions taking place and structures of the compounds formed; FIG. 75A-C shows HPLC chromatograms collected at each stage and FIG. 76absorption spectra extracted from each peak. Step A) Labeled,3′-O-protected nucleotide shows one peak (1) and absorption at bothnucleotide (280 nn; note the max for the propargyl cpds is shiftedtowards 280-290 nm) and the dye (575 nm). Step B) Treatment with DTTproduces peak 2 with absorption peak of the dye (575 nm) and migratingslower (more hydrophobic) and peak 3 with (278 run) absorption andfaster migration due to more hydrophilic character. Step C) Additionaltreatment with TCEP produces peak 4 with absorption max at 278 andwithout the dye at even lower retention time consistent with the loss ofthe 3′-OH protective group. The cleaved dye splits into additional peak(5, 6) but both peaks have identical absorption.

Another example of cleavage is shown in FIG. 77 and FIG. 78A-B. FIG. 77shows scheme for cleavage reaction using nucleotide carrying dithiobased protective group on the 3′ end and dithio based linker. As thisfigure shows the cleavage reaction could be performed as one step or 2step process. FIG. 78A-B shows results of cleavage experiments performedusing variety of cleavage agents: dithiosuccinic acid, L-cysteine, DTTand cysteamine. FIG. 78 (A) shows RP-HPLC chromatograms generated forstarting material and reaction mixtures after incubation with cleavageagents dithiosuccinic acid, L-cysteine, DTT and cysteamine. FIG. 78 (B)shows identified compositions of reaction mixtures indicating fullcleavage of both linker and the 3′-protective groups in case ofL-cysteine, DTT and cysteamine, and selective cleavage of3′-O-protective group in case of dithiosuccinic acid. This indicatesthat selectivity can be achieved by choosing structures of linker,protecting group and the nature of cleaving agent (i.e., with varyingpKa of the SH groups and degree of steric hindrance). In addition tothese a variety of suitable cleaving agents can be used such asBis(2-mercaptoethyl)sulfone (BMS) andN,N′-dimethyl-N,N′-bis(mercaptoacetyl)hydrazine (DMH) (Singh et al.,Bioorg. Chem., 22, 109-115 (1994) [17]. Reactions can be furthercatalyzed by inclusion of selenols (Singh et al. Anal Biochem. 1995 Nov.20; 232(1):86-91 [18]). Borohydrides, such as sodium borohydrides canalso be used for this purpose (Stahl et al., Anal. Chem., 1957, 29 (1),pp 154-155 [19]) as well as ascorbic acid (Nardai et al., J. Biol. Chem.276, 8825-8828 (2001) [20]). In addition, enzymatic methods for cleavageof disulfide bonds are also well known such as disulfide andthioreductase and can be used with compounds of the present invention(Holmgren et. al., Methods in Enzymology, Volume 252, 1995, Pages199-208 [21]).

Example 81 Scavengers

Accordingly to the cleave agent used one skilled in the art needs tochoose a scavenger agent which will remove excess of cleave agent aftercleavage reaction is completed. For example, for thiol bearing cleaveagents, a scavenger capable of reacting with free SH group can be used.For example, alkylating agents such as iodoacetamide or maleimidederivatives can be used (U.S. Pat. No. 8,623,598 [47], hereinincorporated by reference). For borohydrides, one skilled in the artcould use ketone bearing compounds, for example levulinic acid orsimilar compound. Finally, one could also use oxidizing reagent tooxidixe excess cleave agent to non-reactive species, for exampleperiodate (Molecules 2007, 12(3), 694-702 [48]).

Example 82 Modular Synthesis

Labeled nucleotides of the present invention require several steps ofsynthesis and involve linking variety of dyes to different bases. It isdesirable to be able to perform linker and dye attachment in a modularfashion rather than step by step process. The modular approach involvespre-building of the linker moiety with protecting group on one end andactivated group on the other. Such pre-built linker can then be used tocouple to propargylamine nucleotide, deprotect the masked amine groupand then couple the activated dye. This has the advantage of fewer stepsand higher yield as compare to step-by-step synthesis. For example,Compound 32 in FIG. 13 is an example of preactivated linker comprisingcleavable functionality, with activated reactive group (disuccinimidylcarbonate) and masked/protected amine (Fmoc). After coupling to freeamine on propargylamine nucleotide the protective group can beconveniently removed for example by treatment with base (aq. Ammonia,piperidine) and can be coupled to activated (NHS) dye molecule.

Example 83

Linkers of the present invention were tested to measure theirhydrophobicity. The log P value of a compound, which is the logarithm ofits partition coefficient between n-octanol and waterlog(c_(octanol)/c_(water)) is a well-established measure of thecompound's hydrophilicity (or lack thereof) [49]. Low hydrophilicitiesand therefore high log P values cause poor absorption or permeation. Inthis case, the log P value was calculated using predicitive software,the table below shows the results, indicating that the linkers (such asthose in FIG. 25) are hydrophobic linkers, while some commercially usedlinkers are hydrophilic.

Molecular LogP Linker Formula Osiris* ChemDraw Molinsp. ** MarvinSketchLegacy C8H16N2O2S2 0.60 0.49 −0.14 −0.76 New C22H43N3O8S2 2.57 2.09 1.300.71 ILMN PEG11 C43H74N6O18 −1.80 −1.80 −2.37 −1.30 ILMN PEG23C63H114N6O28 −2.74 −3.60 −4.34 −1.77

Example 84

This example shows the synthesis of 3′-O-(methylmethylenedisulfide)5-(N-triflouroacetyl-aminopropargyl)-dU ((MeSSdU-PA(COCF3), 7). See,FIG. 85.

A. Synthesis of5′-0-(tert-hu0,1dimethylsilyl)-5-(N-trifluoroacetyl-aminopropargyl)-2′-deoxyuridine(2)

5′-O-(tert-butyldimethylsilyI)-5-iodo-2′-deoxyuridine (1, 25.00 g, 53.37mmol) was dissolved in dry DMF (200 mL) and treated withtetrakis(triphenylphosphine)palladium (0) (6.16 g, 5.27 mmol) and CuI(2.316 g, 12.16 mmol) at room temperature for 10 minutes under argonatmosphere. Then N-trifluoroacetyl-propargylamine (23.99 g, 157.8 mmol)and Et₃N (14.7 mL, 105.5 mmol) were added sequentially and stirred for3.0 h at room temperature. Solvent was then removed by rotaryevaporation. The resulting crude product was dissolved in 500 mL EtOAcand transferred into a separating funnel. The organic part was thenwashed with saturated NaHCO₃ (2×400 mL) and saturated NaCl (2×400 mL)solutions, respectively.

The EtOAc part was then dried over anhydrous Na₂SO₄. After filtratingoff the Na₂SO₄ salt, the filtrate ethyl acetate part was concentratedusing a rotary evaporator. It was then purified by a silica gel Flashchromatography (1:1 Hex:EtOAc to 2:3 Hex:EtOAc, 200 g, 15 u HP puriflashcolumn, 3× injection) bonding to 3×40 g silica gel resulting in 21.994 gof 2 (83.88% yield). HR-MS: Obs 492.1773, calcd for C20H29F3N3O6Si[M+11]+492.1699. 1H-NMR in DMF-d7: 6H 11.65 (brs, 1H, NH), 10.15 (brs,1H, NH), 8.15 (brs, 1H), 6.37 (t, J=5.99 Hz), 5.42 (m, 1H), 4.41 (m,1H), 4.37 (brs, 2H, for NH—CH₂ of propargylamine group), 4.00 (m, 1H),3.84-3.97 (m, 2H), 2.30 (m, 1H), 2.20 (m, 1H), 0.97 (s, 9H) and 0.19 (s,6H) ppm. B.

Synthesis of5′-0-(tert-butydimethylsilyl)-3′-0-(inethylthioinethyl)-5-(N-trifluoroacetylaminopropargyl)-2′-deoxyuridine(3)

Compound 2 (21.99 g, 44.77 mmol) obtained in the earlier step wasdissolved in DMSO (90 mL) in a 1000 mL round bottom flask. It was thenadded sequentially with AcOH (40 mL) and acetic anhydride (132 mL) andstirred for 48 hours at room temperature. The reaction mixture wasneutralized by slowly saturated K2CO3 until the evolution of CO2 gas wasceased. The mixture was then transferred into a separating funnel andfurther extracted (2×500 mL CH₂Cl₂). The combined organic part was thenwashed with saturated NaHCO₃ (1×500 mL) and dried over Na2SO4. Theorganic part was purified by silica gel flash chromatography(Hex:EtOAc/7:3 to 1:1) yielding 12.38 g UtaisilfunitS4b§ 0.03/n884alculated for C22H33F3N3O6SSi (M+H)+552.17. 1H-NMR of compound 3(DMSO-d6): 6H 11.69 (s, 1H), 10.01 (s, 1H), 7.93 (s, 1H), 6.07 (m, 1H),4.69 (m, 2H), 4.38 (m, 1H), 4.19 (m, 2H), 4.03 (m, 1H), 3.75 (m, 2H),2.34 (m, 1H), 2.14 (m, 1H), 2.07 (s, 3H), 0.86 (s, 9H) and 0.08 (s, 6H)ppm.

C. Synthesis of 3′-0-(TMPMT)-5′-0-(tert-butyldimethylsilyl)-5-(N-trifluoroacetyl-aminopropargyl)-2′-deoxyuridine (5)

A round bottom flask was charged with compound 3 (3.120 g, 5.66 mmol),30.0 mL dry CH2Cl2, 3-Å molecular sieves (5.0 g) and cyclohexene (0.70mL, 6.9 mmol). The reaction flask was then placed on an ice-bath. Tothis, SO2Cl2 (8.5 mL, 1M in CH2Cl2, 1.5 eq) was added slowly via asyringe, and stirred for 1 hour at 0° C. Next, an extra of 4.0 mL of 1MSO2Cl2 was added and stirred an additional 40 minutes to ensure completeconversion to compound 4. The volatiles were removed under vacuum whilekeeping the temperature close to 10° C. The resulting solid wasre-suspended in 20 mL of dry DMF and kept under a nitrogen atmosphere.

In a separate flask, (2,4,6-trimethoxyphenyl)methanethiol (3.0 g, 14.15mmol) was dissolved in dry DMF (40 mL) under nitrogen atmosphere, andtreated with NaH (566 mg, 60% in oil, 14.15 mM) producing a grey slurry.To this, compound 4 solution was added at once and stirred at roomtemperature for 2.5 hours under nitrogen atmosphere. The reactionmixture was then filtered through Celite®-S (20 g) in a funnel elutingthe product with EtOAc (200 mL). The EtOAc solution was then washed withdistilled water (3×200 mL). The EtOAc extract was dried over Na2SO4,concentrated by rotary evaporation, and purified by flash chromatography(column: 120 g RediSepRfGold, gradient: 7:3 Hex:EtOAc to 3:7 Hex:EtOAc).The target compound (5) was obtained as white solid (1.43 g, 35.5%yield, R_(f): 0.5, Hex:EtOAc/1:1). 1H NMR (CDCl3): δH 7.98 (m, 1H), 6.09(m, 1H), 6.00 (m, 2H), 4.67-4.51 (m, 2H), 4.30 (m, 111), 4.22 (m, 2H),4.00 (m, 1H), 3.80-3-60 (m, 11H), 2.31 (m, 1H), 1.83 (m, 1H), 0.80 (m,9H) and 0.01 (m, 6H) ppm.

D. Synthesis of3′-O-(methylmethylenedisulfide)-5′49-(tert-butyldimethylsi/lyl-5-(N-trifluoroacetylaminopropargyl)-2′-deoxyuridine(6)

Compound 5 (1.43 g 1.99 mmol) was dried under high vacuum with P2O5 in adesiccator overnight and dissolved in 25 mL of anhydrous CH₂Cl₂ in aflask equipped with a magnetic stirrer and nitrogen gas source. To thiswas added dimethyldisulfide (0.89 mL, 9.88 mmol), and the reaction flaskwas stirred on an ice-bath. Dimethyl(methylthio)sulfoniumtetrafluoroborate (DMTSF, 430 mg, 2.19 mmol) was then added and stirredfor 1.0 hours at 0° C. The reaction mixture was transferred to a 500 mLseparatory funnel and quenched with 100 mL of 50 mM aq. solution ofNaHCO₃, and extracted with CH₂Cl₂ (2×150 mL). The organic portion wasdried over Na2SO4 and concentrated by rotary evaporation. The crudeproduct was purified on a silica gel column (80 g RediSepRf gold) usinggradient 8:2/Hex:EtOAc to to 3:7/Hex-EtOAc to result in 0.622 g ofcompound 6 (54% yield, RF=0.6, Hex:EtOAc/1:1). 1H NMR (CDCl3): 811 7.99(brs, 1H, NH), 7.98 (s, 1H), 6.12 (m, 1H), 4.69 (m, 2H), 4.35 (m, 1H),4.19 (m, 2H), 4.06 (m, 1H), 3.80 (m, 1H), 3.60 (m, 2H), 2.40 (m, 1H),2.33 (s, 3H), 1.88 (m, 1H), 0.78 (m, 9H), and 0.10 (m, 6H) ppm.

E. Synthesis of3′-O-(methylmethylenedisulfide)-5-(N-trifluoroacetyl-amino-propargyl)-2′deoxyuridine (7)

A round bottomed flask equipped with a magnetic stirrer was charged withcompound 6 (0.623 g, 1.06 mmol, vacuum dried with P2O5 overnight) andanhydrous THE (20.0 mL) and placed on an ice-bath under a nitrogenatmosphere. TBAF (1.27 mL, 1.27 mmol, in 1M solution) was added slowlyvia syringe. The reaction mixture was stirred for 1.5 hours at 0° C.Then, an extra of 0.9 mL 1M TBAF was added and stirred a total of 4hours at ice-cold temperature. The reaction mixture was then transferredto a separatory funnel and quenched with 0.5 M NaHCO₃ solution (50 mL).The resulting mixture was extracted with EtOAc (2×100 mL) and dried overNa2SO4. The product 7 was obtained as a white powder after silica gelcolumn chromatography in 63% yield (311 mg) on a 80 g RediSepRf columnusing gradient 7:3 to 3:7 Hex:EtOAc. 1H NMR (MeOH-d4): 611 8.16 (s, 1H),6.06 (m, 1H), 4.79 (m, 2H), 4.69 (m, 1H), 4.40 (m, 1H), 4.14 (m, 2H),3.99 (m, 1H), 3.63 (m, 2H), 2.36 (m, 3H), 2.32 (m, 1H), and 2.08 (m, 1H)ppm. Further confirmed by LC-MS: M−H observed m/z 468.0.

Example 85

This example shows the synthesis of3′-O-(methylinethylenedisulfide)-5-(aminopropargyl)-2′-deoxyuridine(MeSSdUTPPA,8). See, FIG. 86. Vacuum-dried sample of compound 7 (155 mg,330 nmol) and proton sponge (100 mg) were placed in a 25 mL pear-shapedflask containing a magnetic stirring bar under Ar-atmosphere. The solidswere suspended in trimethylphosphate (1.4 mL) and stirred for—30 minutesat room temperature until all solids were completely dissolved. Theflask was then placed in a ice-salt water bath and stirred for 10minutes to bring the flask to sub-zero temperature (−5 to 0° C.). ThenPOC13 (50 [IL, 570 nmol) was added at once using a microsyringe. Themixture was then stirred at the same temperature for 1 hour. Next, apremix of pyrophosphate-Bu3N-DMF was prepared as quickly as possible ina 15 mL centrifuge tube producing a thick solution(pyrophosphate-Bu3NH+0.73 g, Bu3N 0.73 mL, dry DMF 2.6 mL). Oncecompletely dissolved, the mixture was rapidly added to the rigorouslystirring reaction mixture at once. The reaction mixture was stirred atroom temperature for 15 mins. The reaction mixture was then poured into200 mL of 0.1M TEAB buffer in a 500 mL round bottom evaporation flask.The mixture was stirred for 3 hours at room temperature. The reactionmixture was then treated with 60 mL of ammonium hydroxide (28-30% NH3content) for 1 h at room temperature. The mixture was then concentratedby rotary evaporation and purified by prep—HPLC (19×250 mm, C18 Sunfire,Waters, method: 0 to 2 min 100% A, followed by 20% B over 70 mins, flow14 mL/min; A=50 mM TEAB, B=ACN). The target fractions (Rf=32-37 mins)were lyophilized and combined after dissolving in HPLC grade water toresult in 103 nmol of MeSSdUTP-PA, 8 (58% yield). The product wasconfirmed by LC-MS m/z (M−H) 612.00.

Example 86

This example shows the synthesis of Synthesis of MeSSdUTP-ARA-NH2, 11:

Compound MeSSdUTP-PA (8) was aliquoted in a 15 mL centrifuge tube (2.16mL @6.92 mM=15.tmol). It was diluted with 0.8 mL HPLC grade water and1.5 mL of freshly prepared 0.5 M Na2HPO4 in HPLC grade water. In aseparate tube, 35 mg of activated linker NHS-ARA-Fmoc (10, 44 f.imol)was suspended in 3.0 mL dry DMF. This solution was was added to theMeSSdUTP-PA/Na2HPO4 solution in once and gently shaken by hand. It wasthen placed on shaker and allowed to react for overnight at roomtemperature. Next day, HPLC analysis showed quantitative conversion toconjugated product (10). It was diluted with 1.0 mL of 0.1M TEAB buffer.To this, 0.7 mL of piperidine was added and stirred on a shaker for 1hour at room temperature. The product was then immediately purified byprep HPLC on 30×250 mm C18 Sunfire—Waters column, method: 0 to 2.0 min100% A, followed by 50% B over 70 min, flow rate: 25 mL/min, A=50 mMTEAB, B=acetonitrile, 1 injection only. The target fractions (Rf=46 min)were combined and lyopphilized, and final product was dissolved in HPLCgrade water and the concnetration was determined by UV-spectrophotometryto result in 6.8 nmol of MeSSdUTPARA-NI-12, 11 (45% yield in two steps).The product was further confirmed by LR-LC-MS, observed m/z 1067.

Example 87

This example presents a general method for cleavage studies as presentedherein.

A stock solution of 100 mM cleave reagent (see Table-1) was prepared in1.0 M TE buffer (pH 8.5, Sigma #T9285) in a 15.0 mL centrifuge tube.Then an aliquot of 20 μL (2,000 nmol) of it was diluted with 76.9 uLdistilled water in a 1.0 mL centrifuge tube and added with 3.07 μL of6.5 mM MeSSdUTP-ARA-NH2 (11, 20 nmol) to obtain—100 litL of total volume(Final concentration: 20 mM cleaving agent, 0.2 mM nucleotide, 200 mM TEbuffer). The mixture tube was heated at 65 degree C. on a pre-heatedthermocycler by shaking gently. The mixture was immediately analyzed byLC-MS after 10 minutes. The % conversion was determined by relative peakareas of the respective peaks extracted at 280 mu wavelength.

Example 88

In this example, a comparison is made in DNA sequencing under twodifferent conditions/configurations. In one configuration (B290), acompound is used as a sequencing additive, i.e.5-Chloro-2-methyl-4-isothiazolin-3-one is utilized in the extend reagentfor the extension reaction. In the other configuration (B233), thiscompound is not utilized in the extend reagent for the extensionreaction. As can be seen from FIG. 96, a significant improvement in readlength is observed for the B290 configuration (157 cycles) as comparedto the B233 configuration (137 cycles). As can be seen from the FIG. 97,a significant improvement in read quality (reduced degradation from theinlet to the outlet region of the flow cell) is observed for the B290configuration (157 cycles) as compared to the B233 configuration (137cycles).

Although the invention has been described with reference to thesepreferred embodiments, other embodiments can achieve the same results.Variations and modifications of the present invention will be obvious tothose skilled in the art and it is intended to cover in the appendedclaims all such modifications and equivalents. The entire disclosures ofall applications, patents, and publications cited above, and of thecorresponding application are hereby incorporated by reference.

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1-30. (canceled)
 31. A method for detecting labeled nucleotides in a DNAsequence comprising the steps of a) providing i) nucleic acid templateand primer capable of hybridizing to said template so as to form aprimer/template hybridization complex, ii) an extend reagent comprisingpolymerase, a plurality of nucleotide analogues, and5-Chloro-2-methyl-4-isothiazolin-3-one, wherein said nucleotideanalogues comprise a nucleobase and a sugar, said sugar comprising acleavable protecting group on the 3′-O, wherein said cleavableprotecting group comprises a disulfide-based 3′-terminator group, andwherein at least a portion of said nucleotide analogues furthercomprises a first detectable label attached via a cleavabledisulfide-containing linker to the nucleobase; iii) a cleave reagentcomprising a thiol-containing compound; iv) a cleave scavenger reagent;and v) a flow cell; b) hybridizing at least a portion of said primers toat least a portion of said template molecules so as to create hybridizedprimers; c) exposing said hybridized primers to said extend reagent inan extension reaction in said flow cell under conditions such that afirst nucleotide analogue comprising a first detectable label isincorporated into at least a portion of said hybridized primers in apolymerase catalyzed primer extension reaction so as to create extendedprimers comprising an incorporated nucleotide analogue in a modifiedprimer/template hybridization complex, wherein5-Chloro-2-methyl-4-isothiazolin-3-one inhibits cleavage of saiddisulfide-based 3′-terminator group during the extension reaction; d)detecting said first detectable label of said deoxynucleosidetriphosphate in said modified primer/template hybridization complex; e)introducing said cleave reagent under conditions so as to remove saidcleavable protecting group and said detectable label from said modifiedprimer/template hybridization complex; and f) introducing said cleavescavenger reagent so as to scavenge any residual cleave reagent.
 32. Themethod of claim 31, wherein said detecting is by imaging.
 33. The methodof claim 31, said detecting allows for the determination of thenucleobase of said incorporated first deoxynucleoside triphosphate. 34.The method of claim 31, wherein the method further comprisesincorporating a second nucleotide analogue comprising a seconddetectable label during a repeat of step c).
 35. The method of claim 31,wherein the thiol-containing compound is a vicinal dithiol-basedcompound.
 36. The method of claim 35, wherein the vicinal dithiol-basedcompound is di-mercaptopropanesulfonate.
 37. The method of claim 34,wherein the nucleobase of said second nucleotide analogue is differentfrom the nucleobase of said first nucleotide analogue.
 38. The method ofclaim 31, wherein said extend reagent comprises a mixture of at least 4differently labeled nucleotide analogues representing analogs of A, G, Cand T or U.
 39. The method of claim 31, wherein said detecting allowsfor the determination of the nucleobase of said incorporated firstnucleotide analogue.
 40. The method of claim 31, wherein saiddisulfide-based 3′-terminator group comprises methylenedisulfide as acleavable protecting group.
 41. The method of claim 31, wherein saidextend reagent further comprises 2-Methyl-4-isothiazolin-3-one.
 42. Themethod of claim 31, wherein said scavenger is cystamine.
 43. The methodof claim 31, wherein said flow cell is in fluidic communication with afirst reservoir comprising a cleave reagent comprising athiol-containing compound and a second reservoir comprising an oxidativewash.
 44. The method of claim 43, wherein said oxidative wash compriseshydrogen peroxide.
 45. The method of claim 43, wherein said oxidativewash comprises ter-butyl peroxide.